Name: imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique
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Description: Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

倫理文：動物を対象とするすべての手順は、ニューサウスウェールズ大学の動物管理及び倫理委員会（ACEC）（承認ACEC番号は14 / 94Aである）のガイドラインに従ってください。 透明なマウス脊髄の調製<ol><li>アイスコールドヒドロゲル溶液の調製<ol><li> 16％パラホルムアルデヒド溶液（PFA）の調製<ol><li> 70ミリリットル予め温めておいた蒸留水（50〜55℃）でに16グラムのパラホルムアルデヒド粉末を追加し、パラホルムアルデヒドが溶解するまで加熱された磁気撹拌機で撹拌します。注：ソリューションがオーバー55°Cを加熱し、パラホルムアルデヒドは有毒であることに注意することがないようにしてください。 </li><li>シリンダーにパラホルムアルデヒド溶液を移し、100mlに蒸留水を追加します。 4℃の冷蔵庫を使用して、パラホルムアルデヒド溶液を冷却します。 </li></ol></li><li>蒸留水26.25ミリリットル中に125mgのVA-044開始剤を溶解させ、4溶液を冷却Cの冷蔵庫を°。 </li><li>クール5ミリリットルアクリルアミド溶液（40％）、1.25ミリリットルビス溶液（2％）（注意：ビスアクリルアミド溶液は毒性があり、遺伝的欠陥や癌を引き起こす可能性がある）、5ミリリットル10×PBS、12.5ミリリットル16％PFA溶液、および氷上のVA-044の開始剤溶液。 </li><li>氷の上に上記の溶液を混合。 注：総容量は50ミリリットルです。 </li></ol></li><li>マウス脊髄の灌流およびコレクション<ol><li> 0.9％生理食塩水で希釈したケタミン（80mg / kg）およびxyzaline（5mg / kg）の腹腔内注射でマウスをAnaesthetize。マウスのための用量は、ラットのそれよりも低くすることができます。 </li><li>マウスは、足の皮にピンチに応答しません一度ヒュームフード内で適切なプラスチック表面に、（粘着テープが効果的である）身体から離し、マウスの四肢を固定します。 </li><li>胸の上にマウスの皮膚をカットした後、骨の胸は、はさみで心臓を露出します。 </li><li> 25 G針が番目に取り付けると、蠕動ポンプを使用してチューブの電子端部は、心臓の左心室に針を挿入し、約10ミリリットル/分の速度を血液のための出口点を作成し、40ミリリットルの0.9％生理食塩水での洗浄を開始するために右心房を切り取ります。 </li><li>完了したら、35ミリリットルの氷冷ヒドロゲル溶液でマウスを灌流。 注：神経組織の膨張を回避するために、10ml /分の速度で灌流。 </li><li>ブレードバックの上に皮膚を切開して、次の脊椎動物に筋肉を削除します。一方の側で椎弓を切断し、反対側にフリップした後、細かいハサミで脊柱から脊髄を取ります。 </li></ol></li><li>マウスの脊髄をクリア<ol><li> 4℃で一晩ヒドロゲル溶液の15ミリリットルで脊髄を置きます。 </li><li>ヒドロゲル溶液の10ミリリットルを削除し、5ミリリットルチューブに脊髄セグメントを有する液の残りを注ぎます。それは完全に一杯になるまでヒドロゲル溶液で5 mlのチューブを埋めます。 </li><li> 5ミリリットルチューブの上にパラフィルムの小片をストレッチした後、チューブの首にパラフィルム包みます。注：パラフィルムの接点ヒドロゲル溶液を確認し、ヒドロゲル溶液と、パラフィルムの間に気泡が存在しません。 </li><li> 37で5ミリリットルチューブを入れて&#160; °Cのオーブンで一晩。それがゲルになるまでヒドロゲル溶液を観察します。 </li><li>オーブンから5ミリリットルチューブを取り、（そのようなシャベル1ミリリットルピペットチップなど）スパナでチューブからゲルを削除します。ゲルに粗い組織を貼り付けることにより、脊髄組織からゲルを外し、（ゲルは組織に固執するため、組織によって削除されます）離れ粗い組織を取ります。 </li><li>脊髄組織からゲルを除去した後、シェーカー上で24時間、1×PBS（pH7.4）で脊髄を4回洗浄します。 </li><li> 3.85グラムの尿素3.85グラムのN、N、N &#39;、N&#39;-テトラキス（2-ヒドロキシプロピル）ethylenediamを溶解することによりキュービック透明溶液を調製します5.38ミリリットルの蒸留水にINE。 注：ホットスターラーが使用されています。それは明確であり、室温にまで冷却後に溶液に、2.31グラムのポリエチレングリコールモノ-P-イソオクチルフェニルエーテル/トリトンX-100を追加します。 注：これらの3化学物質の10グラムは1マウス用です。 </li><li>かみそりの刃で2〜3ミリメートル長いセグメントに冠状にマウスの脊髄を切り取り、上記CUBICクリア溶液5mlに入れて。 37のオーブンでシェーカー上に置き&#160; 3日間Cを°。 </li><li> 3日後、新鮮なものに決済ソリューションを変更します。注：上記溶液は使用前に調製されます。 </li><li> 2〜3日後にCUBIC決済ソリューションをリフレッシュした後、フォント8文字で紙に対する組織の透明性を確認してください。それが透明である場合、文字がクリア組織を通して見ることができます。 （0.1％のTriton-X100）は、組織を1日4回（6時間ごと）を洗浄するためにクリア溶液を除去し、PBSTで4ミリリットルを追加します。 </li></ol></li></ol><ol><li> 37℃のオーブンでシェーカー上で3日間：一次抗体溶液中の脊髄セグメントをインキュベート（PBSTで100抗セロトニンは、ウサギで育った、1に希釈しました）。 </li><li>抗体溶液を除去し、37℃のオーブンで脊髄セグメントを1日4回（各6時間）を洗浄するためにPBST（0.1％のTriton-X100）の4ミリリットルを追加します。 </li><li> PBST溶液を除去し、二次抗体溶液を加える（594結合ヤギ抗ウサギIgG、1希釈：PBSTで100）を37℃のオーブン中でシェーカー上で3日間組織をインキュベートします。 </li><li>二次抗体溶液を除去し、37℃のオーブンでシェーカー上脊髄セグメントを1日4回（6時間ごと）を洗浄するためにPBSTの4ミリリットルを追加します。翌日、組織は、イメージングのための準備ができています。 </li></ol> 3.イメージング<ol><li> PBST溶液を除去し、さらには組織の屈折率を作るために85％グリセロールの4ミリリットルを追加します。 </li><li&gt;は組織の透明性を確認してください。それは約1時間で明らかになると、マルチフォト蛍光顕微鏡の保持枠に適合する大、薄いガラススライド上に組織を置きます。 </li><li>次の脊髄組織に85％グリセロールの数滴を入れて、22×50mmの2カバースリップガラスでそれをカバースリップ。 注意：脊髄の向きは、画像がどのように見えるかを決定します。 </li><li>多光子蛍光顕微鏡の保持枠の上にスライドガラスを入れて、光経路に組織を動かします。 </li><li>ヘリウムネオンレーザライン594 nmのを選択して、ライブ画像に正の信号の輝度をチェックして、最適なレベルにレーザーの強度を調整します。 </li><li> （水中、NA 0.7）20Xの対物レンズを用いて脊髄組織の走査領域を選択し、zスタックを作成するための準備、および3μmと各ステップの深さ。 </li><li> 20XのOBJEC下zスタックの上から下に組織をスキャン的と（油中、NA 1.4）を63Xの対物レンズをそれぞれ、（ステップサイズは1μmでした）。 3Dソフトウェア28を使用して3D映像を再構成します。 </li></ol>

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The authors have nothing to disclose.

プロトコルを組み合わせCLARITYとCUBIC技術を用いてマウスの脊髄における画像セロトニン作動性繊維にどのようにショーを説明しました。これは、チャンら 14およびさまら 15によって開発された受動的な決済プロトコルに比べて速くクリア処理を紹介し、脊髄組織が ​​うまくクリア時にヒドロゲルによってサポートすることができます。 チャンら 14およびさまらによって報告されているように、マウスの脊髄の固定、中に重要なステップ。15は 、化学物質の重合を防止する、すべての溶液を氷上で冷静さを保つことです。他の重要なステップは、神経組織の膨潤を回避するために、ヒドロゲル溶液でゆっくりとマウスを灌流することです。ヒドロゲル溶液がゲルになるためには、脱気が必要とされます。これに代わるものとして、我々は、チューブの上部をカバーするためにパラフィルムを使用し、HYDとの間に気泡が残っていませんrogelソリューションとパラフィルム。これは非常によく働いたとヒドロゲル溶液は、数時間後にゲルになりました。いくつかの気泡がゲル中に見出されたが、それらは、脊髄組織中の気泡が存在しないことによって証明された以下の実験を妨害しませんでした。クリアは、最も重要なステップであり、および試薬の組み合わせを必要とします。アミノアルコールを含む決済ソリューションは、元CLARITYプロトコル16で使用されるSDS /ホウ酸系決済ソリューションよりもはるかに高速脊髄組織の脂質を除去します。 伝統的な免疫組織化学技術上CLARITY立方の利点は、全体の組織ブロックは、セロトニン作動性線維の連続性が保持され、その結果、組織を切片化することなく画像化することができ、長い距離を同じファイバに追従することが可能であることですzスタック内。この利点により、担保は自信を持って3D画像で識別することができ、ND新規な結論は、したがってセロトニン経路の性質について行うことができます。記載されているプロトコルは、単純に特異的な抗体で組織を標識することにより、ファイバシステム、ならびに核の調査のための便利なツールです。この技術は、二重または三重標識を使用して、同じマウス脳内の異なるシステムを問い合わせるための理想的な選択肢です。我々の研究室の設定では、多光子蛍光顕微鏡は、300μm程度の作動距離で使用可能でした。組織の両面をスキャンしている場合、これは600ミクロンの最大値に私たちの画像を制限します。しかし、光シート蛍光顕微鏡、その全幅、長さと深さを介して、画像全体マウス脳と脊髄をすることができます。この技術の他の利点は、抗体が溶出することができるし、別の異なる抗体または抗体が同じ組織に再び適用されるということです。これは動物に使用かなりだけでなく、新たな脳組織の準備に関連するコストを削減し各実験について。同じ理由で、この技術は、他の組織にも適用することができます。すでに肺、腎臓、心臓、腸、および腫瘍組織26,27に実施された研究が行われています。 また、この手法には限界があり、例えば、マウス後脳内縫線核は、GABA作動性ニューロンを含むニューロンの多くの種類が含まれています。 CLARITYとCUBICは、単一のマウス脳と脊髄でraphespinal繊維の多様な種類の分布の研究を可能にしますが。彼らの神経伝達物質またはタンパク質マーカーにより具体的に特定されていないこれらのニューロンは、この設定では識別できません。代替は、in situハイブリダイゼーションのような核酸プローブと特定のニューロンまたはファイバシステムにラベルを付けることです。これは、一方で、ラベリングシステムと蛍光イメージングのために使用可能なフィルタによって制限されます。したがって、すべてのraphespinal繊維を画像化することは課題のまま。これらの技術は順行性トレーサーの頭蓋内注射と一緒にした場合（例えば、インゲンマメのleucoagglutininとして - PHA-L）は、セロトニン作動性繊維、GABA作動性繊維、および他の人を見ることは可能かもしれません。

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table><tbody><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>40% acrylamide solution</td><td>Bio Rad</td><td>161-0140</td><td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td></tr><tr><td>2% Bis Solution</td><td>Bio Rad</td><td>161-0142</td><td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-&lt;br/&gt;879c-4d63-ba0b-82556a0ccf1d</td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>158127</td><td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&amp;amp;region=AU</td></tr><tr><td>urea</td><td>Merck Millipore</td><td>66612</td><td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#039;,N&#039;-tetra-2-propanol,MDA_CHEM-821940</td></tr><tr><td>Triton-X 100</td><td>Merck Millipore</td><td>648462</td><td>http://www.merckmillipore.com/AU/en/product/TRITON&amp;reg;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&amp;amp;region=AU</td></tr><tr><td>serotonin antibody</td><td>Merck Millipore</td><td>AB938</td><td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td></tr><tr><td>goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 594 conjugate</td><td>Life Technologies</td><td>&amp;nbsp;A-11012</td><td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>multi-photon microscope</td><td>Leica</td><td>Leica TCS SP5 MP STED</td><td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td></tr></tbody></table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

脊髄への長い下降繊維が移動、疼痛知覚、およびその他の動作のために不可欠です。これらのファイバシステムの大部分の脊髄における繊維の終端パターンを徹底的に任意の種に調べられていません。脊髄に突出セロトニン作動性線維は、組織切片のラットおよびオポッサムに研究されており、それらの機能的意義は、脊髄でのファイバ終端パターンに推定基づいています。 CLARITY立方技術の発展に伴い、このファイバシステムとセロトニン脊柱上の経路の未知の機能を明らかにする可能性がある脊髄におけるその分布を調べることが可能です。ここでは、合成CLARITY立方技術を使用して、マウスの脊髄におけるセロトニン線維を撮像するための詳細なプロトコルを提供します。この方法は、COMBINと組織のヒドロゲル溶液と明確化とマウスの灌流を伴います試薬をクリアするエーション。脊髄組織は、すぐ下に2週間でクリアされた、およびセロトニンに対するその後の免疫染色は、10日以内に完了しました。多光子蛍光顕微鏡を用いて、組織を走査し、3次元画像は、OsiriXのソフトウェアを使用して再構築しました。

このセクションでは、CLARITY立方プロトコルの組み合わせを使用して、透明なマウスの脊髄におけるセロトニン抗体染色の結果を示します。私たちは、セロトニン作動性繊維は（もビデオ1参照、 図1）前角の腹の部分で優勢で脊髄の全ての薄層に存在していることを示しています。対照組織は陽性線維（結果は表示されませんでした）を持っていませんでした。前角では、高密度に充填されたセロトニン作動性繊維は前角の腹内側部分に存在していると、彼らは前角の外側部に向かって延びている（ 図2、またビデオ2を参照）。いくつかの免疫陽性繊維はまた、背角または中心管に向かって前角から延びています。免疫陽性繊維は、後角の全ての薄層中に存在しているが、正の薄層2中の繊維と4の間に小さな隙間があり、特に横で脊髄後角の一部。後角におけるセロトニン線維の大部分は小さい直径のものであり、それらのほんの数が大径（ ビデオ1）です。水平断面において、前角における密集セロトニン作動性線維は、脊髄の長手方向軸に沿って移動し、大きな繊維束を形成することが観察されました。最も興味深い発見は、この大規模な繊維束は、繊維束に対して垂直であり、定期的に縦軸に沿った枝を、発行していることです。これらの分岐は、さらに前角の外側部分にそのパスに沿って担保します。これらのブランチと比較し、正中線に向かって延在するものが不規則小さい（ 図2）です。 63X対物レンズの下に、後角におけるセロトニン線維は、脊髄の軸に沿って移動し、管の経路に沿った様々な点で終端することが観察されました。太い繊維が散発的に薄いFの間に分散​​されています<html> ibers（ 図3、またビデオ3を参照してください）。 <img alt="図1" src="/files/ftp_upload/53673/53673fig1.jpg" /> 図1：200X倍率左の腰のコードの冠状断面におけるセロトニン作動性繊維は、右後角は、背側脊髄の内側部分であり、および腹側脊髄の横方向の一部であり、前角です。スケールバーは100μmです。セロトニン作動性繊維、特に繊維密度が高い前角の腹内側部に、すべての薄層中に存在します。これらの繊維は、前角の外側部と中央運河や脊髄後角の両方に向かって延びています。他の薄層内の繊維の密度は低いです。セロトニン作動性線維の大部分は薄いです。繊維のみの小さな数が厚くであり、彼らは脊髄後角および前角（両方で見られます&gt;また、ビデオ1参照）（右クリックしてダウンロード）。 <img alt="図2" src="/files/ftp_upload/53673/53673fig2.jpg" /> 図2：腹200X倍率左で前角の部分と右側にあるセロトニン線維は、脊髄の側面であり、背側は、脊髄の吻側一部であり、腹側脊髄の尾の部分です。スケールバーは100μmです。セロトニン作動性線維は、脊髄の縦軸に沿って移動する図示し、それらが厚い繊維束を形成する前角の中央部に充填されています。正中線に向かって延びている他の人が不規則と短いのに対し、このバンドルのうち、いくつかの枝は、前角の側部に向けて定期的に拡張されています。これらの繊維は、（管の経路に沿った多くのポイントで終端<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53673/Video2.mp4">もビデオ2を参照してください。</a>）（右クリックしてダウンロード）。 <img alt="図3" src="/files/ftp_upload/53673/53673fig3.jpg" /> 図3：630Xの倍率で下の脊髄後角におけるセロトニン作動性線維左、右、脊髄、背側と腹側のそれぞれ、背側と後角の腹の部分を指すの内側部分があり、脊髄の横方向の一部です。 。スケールバーは7μmです。セロトニン作動性線維は、脊髄の長手方向軸に沿って移動し、管の経路に沿って終端しています。これらの繊維は、混在太い繊維数の少ない主に小径のです。これらの繊維はほとんど重なっていない（ <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53673/Video3.mp4">また、ビデオ3（右クリックしてダウンロード）を参照してください</a> ）。

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

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

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-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.4K ビュー.  Neuroscience Research Australia. 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.

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

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 ...

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.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

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 ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

パッシブCLARITYを使用したマウス中枢神経系の光学クリア

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

CLARITY組織クリアリング法による全マウントレティナの免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

CLARITY組織クリアリングによるインタクトニューロンデンドライトのイメージングと定量化

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

メゾスコピックから顕微鏡スケールまでのニューロンイメージングのための組織クリアリング法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

低侵襲法に基づくマウス挫傷脊髄損傷モデルの確立

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

C57BL/6Jマウスにおける中枢性臍帯症候群モデルの確立

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...