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...

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.

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			<td height="22" style="height:22px;width:495px;">Photoinitiator VA044</td>
			<td style="width:495px;">Wako</td>
			<td style="width:284px;">va-044/225-02111</td>
			<td style="width:807px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
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			<td height="21" style="height:21px;">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>
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			<td height="21" style="height:21px;">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=5e7a4f31-879c-4d63-ba0b-82556a0ccf1d</td>
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			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td align="right">158127</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&#38;region=AU</td>
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			<td height="21" style="height:21px;">urea</td>
			<td>Merck Millipore</td>
			<td align="right">66612</td>
			<td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td>
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			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td>Merck Millipore</td>
			<td align="right">821940</td>
			<td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#39;,N&#39;-tetra-2-propanol,MDA_CHEM-821940</td>
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			<td height="21" style="height:21px;">Triton-X 100</td>
			<td>Merck Millipore</td>
			<td align="right">648462</td>
			<td>http://www.merckmillipore.com/AU/en/product/TRITON&#174;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td>
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			<td height="21" style="height:21px;">sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&#38;region=AU</td>
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			<td height="21" style="height:21px;">2,2&#8217;,2&#8217;&#8217;- nitrilotriethanol</td>
			<td>Merck Millipore</td>
			<td align="right">137002</td>
			<td>http://www.merckmillipore.com/AU/en/product/Triethanolamine-(Trolamine),MDA_CHEM-137022</td>
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			<td height="21" style="height:21px;">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>
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			<td height="21" style="height:21px;">goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 594 conjugate</td>
			<td>Life Technologies</td>
			<td>&#160;A-11012</td>
			<td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td>
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			<td height="21" style="height:21px;">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>
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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?...

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

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

神経科学

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

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 ...

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 ...

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 ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

慢性脊髄圧迫における拡散テンソル磁気共鳴画像

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

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 ...

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

要約

さらに動画を探す

この動画の章

脊髄電気生理学II：細胞外吸引電極の作製

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

幼虫のゼブラフィッシュにおける脊髄切断

縦断のために脊髄商工を移植するための手順

ラット頸部脊髄の拡散イメージング

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

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

マウスにおける線込みおよび脊髄窓移植

慢性脊髄圧迫における拡散テンソル磁気共鳴画像

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

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

要約

さらに動画を探す

この動画の章

脊髄電気生理学II：細胞外吸引電極の作製

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

幼虫のゼブラフィッシュにおける脊髄切断

縦断のために脊髄商工を移植するための手順

ラット頸部脊髄の拡散イメージング

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

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

マウスにおける線込みおよび脊髄窓移植

慢性脊髄圧迫における拡散テンソル磁気共鳴画像

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

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