Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

このプロトコルで使用されるすべての材料については、表1を参照してください。 実験は、テンプル大学の施設内動物管理使用委員会によって承認された。 1。動物<ol><li>野生型幼虫の海ヤツメウナギ（Petromyzonマリナス L.）を取得し、10 -メイン州のデラウェア川（ペンシルベニア州）またはストリームへの支流から、ミシガン湖の供給流れから- （7歳4）長さが14センチ。 </li><li> 16℃で50ガロンの淡水タンク内の100の動物 - 研究室では、50のグループで幼虫を維持する。ライン砂利のインチでタンク幼虫が中に穴を掘るためにのために、水を炭濾過し、その中にヤツメウナギを置く前に、塩素や他の不純物を除去するために、少なくとも48時間、エアストーンで調整されている。使用する日まで、自らの残骸をリサイクル動物を供給する必要はありません。 </li></ol> 2コンプリート脊髄コー​​ド離断<ol><li>脊髄手術の前に以下の組成を有するヤツメウナギリンゲル液の準備：110のNaCl、2.1のKCl、2.6のCaCl 2、1.8mMのMgCl 2および10mMのトリス緩衝液を; pHは7.4。 </li><li>脊髄切断を行うために、小さな容器に幼虫を置き氷冷リンゲル液中の1％トリカインメタンスルホネートで麻酔する。 </li><li>麻酔された場所にペトリ皿の動物の半分シルガード（シリコーン）で満たし、一動物の鼻に配置される（手術のための位置に尾少なくとも一つを、動物を保持するために4昆虫ピン（0.15ミリメートルの直径）を使用して一度そして2つの5 番目の鰓のレベルで）。ほとんどリンゲル液でトップまでのペトリ皿を埋める。実体顕微鏡下で手術を行いながら、氷上で幼虫をシャーレを入れてください。 </li><li>脊髄離断に背を実行するには、正中線、5 番目のレベルで外科用メス（＃11）と長手方向に切開する脊髄を可視化する鰓。最初に皮膚を上に向けて外科用メスの刃の先端を挿入し、脊髄を上から見ることができるまで、筋肉組織が非常に緩やかに切断されることによって開放されている。ファッション2昆虫ピンからフックと脊髄離断を行いながら、身体の壁が開いて保持するためにそれらを使用しています。 </li><li>完全にハサミを使用して、5 番目の鰓（カストロ＃8）のレベルで脊髄を横断する、脊髄に垂直にはさみを挿入します。脊髄は完全に1きれいにカットを横切する必要があります。脊髄を完全に離断されたことを確認する実体顕微鏡下で脊髄切断端を可視。そして、一対の鉗子（＃5）を用いて、横切開後に脊髄の切断端を再調整。 </li><li>脊髄切断を行った後、微細ピンセットを背体壁に傷を閉じ、創傷tを可能にするために1時間氷上に幼虫を入れO乾燥させます。氷と幼虫の間のフィルタ·一枚の紙を入れて、ヤツメウナギのリンゲル液でそれを浸します。低温は、傷が空気乾燥することができますので、感染を予防するのに役立つと同時に、生きている動物を保持します。 </li><li>その後、24時間氷冷水小さな水槽（タンクあたり1幼虫）の幼虫を移す。 </li><li>行動的に損傷部位への移動、尾がないことを確認するために、脊髄離断後に病変幼虫1日を調べます。幼虫（5 番目の鰓のレベルで）損傷部位にのみ、その頭の吻側を動かすことができれば、脊髄離断が完了したと見なされる。この場合、幼虫は現在、室温で淡水と戦車（タンクあたり1幼虫）に転送することができます（23 それらは、選択された時点で回復することを可能にする°C）。 </li><li>回復期間中は2〜3日ごとに水の交換。 </li></ol> 3。脳解剖との調製in situハイブリダイゼーションのための脳<ol><li>損傷後の希望の時点で幼虫の海ヤツメウナギと場所を再度麻酔し、上記のように、脳の解剖を行うためにリンゲル溶液とペトリ皿で​​それらをピン。 </li><li>脳を解剖するには、最初の鼻孔および松果体臓器の間にメスで横切開を行います。ここからピンセットでそれを保持しながら、カストロのハサミを使用して、脳の周囲の組織を切断し始める。 </li><li>脳が露出された後、ピンセットを使って、脈絡叢を除去し、動物の頭部を広げ、脳神経を露出するためのフックを使用しています。その後、カストロのはさみで脊髄横断を切った。脳を切開する脳神経を切断するために鉗子の別のペアを使用しながら、ピンセットで脊髄を持ちます。 </li><li> 2プラグを挿入することで解剖した後、シリコンの小片に脳を入れて、位置に保持脊髄の嗅球と1電気ショック療法のピン。その後、はさみで背側正中線に沿って後方と脳のcerebrotectal交連をカットし、横方向に翼状プレートを偏向し、4昆虫ピン（ 図2）を有するシリコーンフラットそれらをピン。 </li><li>回転装置上で3時間室温温度に固定されるリン酸緩衝生理食塩水（pH7.4）中4％パラホルムアルデヒドを含むチューブに脳シリコーン片を移す。 </li><li>前述のように定着後のin situハイブリダイゼーションホールマウント用に脳を使用して（スウェインら 、1994）。コントロールまたは偽手術動物からの脳を負傷した動物からの脳と並行して、常に実行する必要があります。 wholemounted幼虫の脳は、そのフラットで薄型のin situハイブリダイゼーションにより研究し、また、脳はかなり半透明になりミエリン10の欠如に起因することができます。 </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

ここでは、幼虫の海のヤツメウナギで完全な脊髄離断および後脳の解剖を実行するために詳細なプロトコルを提示する。この手順では、in situハイブリダイゼーションホールマウント脳による脊髄損傷後のニューロンを投影する識別可能な脊髄の間の遺伝子発現の差を分析することができる。手順における重要なステップはstereomicrocope下、脊髄の切断端を観察することによって制御され、穏や...

哺乳動物では、脊髄損傷（SCI）は、負傷した軸索がトラウマゾーンを通って再生し、それらの適切なターゲットに再接続していないため、損傷部位以下の機能の永久的な損失につながる壊滅的な条件である。哺乳類とは対照的に、ヤツメウナギは、完全な脊髄損傷後の運動を回復。1興味深いことに、ヤツメウナギは、それらの大きなサイズのホールマウント脳調製物において個別に識別可能なニューロンを投影する36脊髄のセットを持っている2,3（ 図1） 。これらの脊髄投射ニューロンのすべてが高レベルの完全な脊髄離断により軸索切断されている。私たちのグループとその他の以前の研究では、他の人は通常、損傷部位を通じて軸索を再生しながらでも、これらのニューロンのいくつかは非常に低い再生能力を示し、SCI後の機能回復の存在下で、（彼らは &quot;悪い再生器を」と考えられている）ことを示している（それらが考慮される「グラムOOD再生器」）。2,3この特性は、ヤツメウナギ面白い脊椎動物モデルは今度にしようとする神経細胞の本質的な再生能力の違いにつながる良い面と悪い蓄冷脊髄投射ニューロン間の遺伝子発現の違いを研究することができます同じ外因環境での軸索を再生。1 このモデルを用いて、私たちは、以前にそれぞれネトリンとRGMの阻害作用を媒介しUNC5 4,5及びネオゲニンなどの軸索ガイダンス分子レセプターの低い再生能力を示して発現とその脊髄投射ニューロン、6を示している。また、この方法を使用することによってわれわれのグループはまた、唯一の良い再生器は、損傷後、再生プロセス中にニューロフィラメントの発現の回復を示すことが示されている。最近では、ブッシュとモーガン7が悪い再生器がincreasを示すことが免疫蛍光によって示されている「悪い再生器「脊髄投射ニューロンはゆっくりと完全な脊髄離断5,7,8の後に死亡するという事実に著者によって関係している傷害後シヌクレインのエド式。だから、完全な脊髄損傷のヤツメウナギモデルは、脊髄に突出ニューロン軸索切断後に「悪い再生器 &quot;を作るかを理解するために非常に有用なモデルとして登場しました。 私たちの研究を実施するために、本 ​​発明者らは、in situハイブリダイゼーションホールマウントを実行するために、損傷した後、所望の時点で完全な脊髄離断手術プロトコルと後方脳の解剖を行っている。現在の方法論的な記事では、幼虫ヤツメウナギにおける完全な脊髄損傷の手術の適正なパフォーマンスのために詳細なプロトコルを提示し、動物のその後のメンテナンスやin situハイブリダイゼーションホールマウントのための最終的な脳の解剖と脳の準備。 PEに詳細なプロトコル幼虫ヤツメウナギの脳においてin situハイブリダイゼーションホールマウントをrformは、以前に報告されている。9はまた、脊髄損傷および脳の解剖のためのこのプロトコルは、その後、免疫組織化学または他の組織学的方法のために脳を処理するために使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="280">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Name of the reagent</td>
			<td style="width:83px;">Company</td>
			<td style="width:64px;">Catalogue number</td>
			<td style="width:71px;">Comments (optional)</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:64px;">Tricaine methane sulfonate</td>
			<td style="width:83px;">Spectrum</td>
			<td style="width:64px;">TR108</td>
			<td style="width:71px;">Benzocaine saturated solution in PBS for sacrifice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Scalpel #3</td>
			<td style="width:83px;">Fine Science Tools (FST)</td>
			<td style="width:64px;">10003-12</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Blades for scalpel: #11</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">10011-00</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Castroviejo scissors #8</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">15002-08</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:64px;">Forceps #4 &#38; #5</td>
			<td style="width:83px;">Dumont, Switzerland</td>
			<td style="width:64px;">Roboz RS4955</td>
			<td style="width:71px;">#4 for dissection of Spinal cord; #5 for stripping menninges</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Dissecting Microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:64px;">SZ51</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:64px;">Sylgard</td>
			<td rowspan="2" style="width:83px;">Dow Corning Co.</td>
			<td rowspan="2" style="width:64px;">184</td>
			<td rowspan="2" style="width:71px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="168">
			<td height="189" rowspan="2" style="height:189px;width:64px;">Insect pins 0.15, 0.20 mm</td>
			<td rowspan="2" style="width:83px;">Austerlitz</td>
			<td rowspan="2" style="width:64px;">No catalogue #</td>
			<td rowspan="2" style="width:71px;">0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">7 ml HDPE Scintillation Tubes with Caps</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td style="width:64px;">03-337-1</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Paraformaldehyde 16%</td>
			<td style="width:83px;">Electron Microscopy Science (EMS)</td>
			<td style="width:64px;">19210</td>
			<td style="width:71px;">Dilute to 4% in PBS</td>
		</tr>
	</tbody>
</table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

完全な脊髄損傷後、最初は海のヤツメウナギは離断のレベル以下に麻痺している。しかし、これらは数週間後に歩行をリカバリし、これを脊髄固有の軸索および脳幹から脊髄に突出する軸索の短い距離再生（数mm）を伴う。 36大の識別可能脊髄投射ニューロンの中には、優れた再生器であり、他は悪い再生器である。これらのニューロンは、最も容易にホールマウントCNS調製物中で同定することができる。有利ニューロン固有のメカニズムを理解または脊椎動物CNSにおける損傷後の軸索再生を阻害するためには、良い、悪い再生器との間の遺伝子発現の相違を決定する方法、および発現は、脊髄切断によって影響される。本稿では、顕微鏡のビジョンの下で、完全な脊髄離断を生産、住宅幼虫のための技術と新鮮な水タンク内の最近変換された大人の海のヤツメウナギを示しており、BRの準備AINおよびin situハイブリダイゼーションのための脊髄wholemounts。簡単に述べると、動物を16に維持される&#160; C°、ヤツメウナギリンガーで1％ベンゾカインで麻酔し。脊髄は背側アプローチを介して、虹彩切除鋏で切開され、動物は23℃の真水タンクに回復させる。 in situハイブリダイゼーションのために、動物を再麻酔され、脳と脊髄は背側アプローチを介して除去。

この方法を使用するときに得られる結果は、識別可能な脊髄突出におけるネオゲニン転写物の発現を示すwholemounted脳の代表画像の一例として、制御のニューロンおよび2週間後の病変幼虫海ヤツメウナギは、 図2に示されている。読者は、完全な脊髄離断および完全な詳細については、海のヤツメウナギの神経細胞を投影する識別可能な脊髄の再生能力の後ネオゲニンの発現との?...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

ヤツメウナギは、完全な脊髄損傷後の運動を回復する。しかし、いくつかの脊髄投射ニューロンが良い再生器であり、そうでないものもあります。本稿では、完全な脊髄離断を生成し、in situハイブリダイゼーションのためのホールマウントの脳や脊髄の準備、住宅海ヤツメウナギの幼生（最近変換された大人）のための手法を例示する。

その後のホールマウントのための完全な脊髄損傷や脳解剖プロトコル<em&gt;その場で</em&gt;幼虫海ヤツメウナギにおけるハイブリダイゼーション

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K ビュー.  University of Edinburgh. ヤツメウナギは、完全な脊髄損傷後の運動を回復する。しかし、いくつかの脊髄投射ニューロンが良い再生器であり、そうでないものもあります。本稿では、完全な脊髄離断を生成し、in situハイブリダイゼーションのためのホールマウントの脳や脊髄の準備、住宅海ヤツメウナギの幼生（最近変換された大人）のための手法を例示する。 

ビデオ: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

二重蛍光その場で</em新鮮な脳切片における>ハイブリダイゼーション

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

横方向の流体のパーカッション：マウスにおける外傷性脳損傷のモデル

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

ゼブラフィッシュ<em&gt;その場での</em&gt;脊髄脊髄感覚運動ニューロンからの電気生理学的記録のための準備

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

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 ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

マウスにおける完全なトランセクション型脊髄損傷の誘導

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...