This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology to M.I. (21229004 and 25221304).

研究のためのラットの使用は、東京大学の動物福祉委員会によって確立されたガイドラインに従いました。 プラスミドDNAの1.Preparation <ol><li>哺乳動物細胞12発現プラスミド中のcDNAまたはshRNA配列をサブクローニングすることによってin vivoでエレクトロポレーションのためのDNAプラスミドを生成します。それは強く、安定した発現を可能にするため、サイトメガロウイルス最初期エンハンサーおよびニワトリβアクチンプロモーターの融合（CAG）プロモーター駆動プラスミド13を使用してください。 CAGプロモーターの制御下でshRNAの発現のために、shRNAの14をサブクローニングのためにmir30ベースのshRNAカセットシステムを使用しています。 </li><li>製造元の指示に従ってマキシプレップキットを用いてプラスミドDNAを精製し、HEPES緩衝生理食塩水（140mMの塩化ナトリウム、0.75ミリモルのNa 2 HPO 4、25mMのHEPES、pHは7.40）でDNAを再懸濁します。 μgの/μLを≥4するDNAの濃度を調整します。 </li><li&gt; 4μgの/μlの濃度にプラスミドDNA溶液を用意し、注射部位を標識するために、高速グリーン染料の最小量（0.01％の最終濃度）を追加します。複数のプラスミドの同時エレクトロポレーションが必要な場合、4μgの/μlのプラスミドDNA溶液の総濃度を調整します。 注：プラスミドDNAの最適組成は、各プラスミドのトランスフェクション効率に応じて決定されるべきです。 </li></ol>手術器具および生理食塩水の2滅菌<ol><li>オートクレーブ手術器具および0.9％NaCl溶液。 </li></ol>ガラスマイクロピペットの調製<ol><li>ピペットプラーを用いてガラスピペットを引き出します。 30-50ミクロンの直径にピペットの先端をカットします。以下のパラメータを使用します。熱、600;ベロシティ、50;時間、75。 </li></ol> 4.動物の手術、DNA注射とエレクトロポレーション注：オーバーこの手順の図は、 図1に記載されている。仔ラットの手順は、ここに記載されているが、この方法は、同様の手順を使用して、より成熟した動物にも適用可能です。 <ol><li>動物は4％（体積/体積）に0.4リットル/分およびイソフルラン濃度に酸素流量を調整することで不動になるまで、インダクションボックス内のイソフルランでラットを麻酔。適切な麻酔を確認するためにピンチつま先を実行します。 </li><li>双眼顕微鏡下で予熱した暖かいにラットを入れ、かつ連続的にフェイスマスクを介してイソフルランを投与することにより麻酔を維持します。 0.2リットル/分および2％（体積/体積）にイソフルラン濃度に酸素流量を調整します。目は動物の目が開いている場合は、目の乾燥を防ぐために廃棄し使用します。 </li><li>外科手術用テープで足を固定してください。 </li><li>ポビドンヨードと後部大腿部の皮膚をきれいにし、メスで切開を行います。 注：外科的領域が時間で覆われている場合は、外科的領域を剃ります空気。 </li><li>大腿四頭筋の間に開口部を作成することによって、坐骨神経を露出すると、筋肉や上腕二​​頭筋が縫い針で筋肉大腿大腿。 </li><li> 0.9％NaCl溶液で神経を濡らします。リントフリーペーパーで余分な水分を吸収します。 </li><li>フレキシブルチューブの上にガラスマイクロピペットの基部を挿入して、静かに吸引することにより、マイクロピペットにDNA溶液（少なくとも1マイクロリットル）の適切な量を記入してください。 </li><li>そっと針を用いて神経の先端側を引っ張ることで露出した神経を持ち上げます。 注意：機械的ストレスを最小限にするために神経に緊張を与えないでください。 </li><li>神経の遠位部位にガラスマイクロピペットを挿入し、（フレキシブルチューブの開放端部に吹き付けることによって）圧力を適用することによって、DNA溶液を注入します。神経が緑（1μlの最大値）が表示されるまでDNA溶液を注入します。マイクロピペットの頻繁な挿入が神経を損傷することがあるので、倍以上のマイクロピペットを挿入しないでください。 </li><li> TWEを配置離れて神経1〜2ミリメートル程度エゼル型白金電極。電極と0.9％NaCl溶液で神経の間のギャップを埋めます。 注：神経への機械的ストレスを避けるために、電極と神経を持たないでください。 </li><li>電極とエレクトロを使用して、注射部位に電気パルスを適用します。最初のパルスセットした後、電極を反転し、別のパルスのセットを適用します。以下のパラメータを使用します。電圧、50 Vを、パルス持続時間、5ミリ秒。パルス間隔、100ミリ秒。パルス数、4回。 </li><li> 0.9％NaCl溶液でエレクトロポレーションサイトを清掃してください。 </li><li>繰り返して、反対側の坐骨神経に4.4から4.11繰り返します。 </li></ol> 5.ポスト​​エレクトロポレーション<ol><li>シアノアクリレート接着剤で切開を閉じます。 </li><li>接着剤を乾燥させた後、ポビドンヨードで傷口をきれいに。 </li><li>フェイスマスクから子犬を解放します。それは完全に麻酔から回復できるようにするために、時間の少なくともウォーマーに子犬を温めます。 Dそれは十分な意識を取り戻したまでO子犬を無人のままにしません。 </li><li>麻酔から回復した後、母ラットに子犬を返します。完全に回復するまでの子犬を返さないでください。 </li></ol> 6.手術後<ol><li>実験11を行うまで、ケージの住宅仔ラット（ 図3の例を参照してください）。カルプロフェン投与（5 mgの/ kgで、腹腔内）、非ステロイド性抗炎症薬、またはブプレノルフィン（0.1ミリグラム/ kg、皮下）、オピオイド鎮痛薬、必要に応じて。 注：ラットの子犬が十分に成長しないか、炎症が手術部位付近に観測されている場合は、実験から動物を除外します。 </li></ol>

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The authors declare that they have no competing financial interests.

In this paper, we describe a simple and efficient method that allows in vivo gene transfer to myelinating SCs in the rat sciatic nerve using electroporation. This method allows highly selective gene expression in myelinating SCs by simply applying electric pulses to the plasmid DNA-injected sciatic nerve. Because the molecular mechanisms of myelination and demyelination in the peripheral nervous system remain unclear, the present in vivo electroporation method will be a powerful tool to clarify the roles of multiple genes of interest in living animals.
A critical requirement of this method is to keep damage to the nerve during surgery to a minimal level. Should surgical damage cause excessive inflammation, the sciatic nerve may degenerate. To avoid this, one must conduct surgery with extreme care, so as to not damage the blood vessels around the nerve. Mechanical stress to the nerve during the surgery can also be a cause of nerve damage. To minimize mechanical stress, lifting the exposed nerve should be done as gently as possible, and the tweezer-type electrode should be placed close to the nerve without contact. Furthermore, electrical pulses that are too strong can cause undesirable large leg movement, which leads to mechanical stress, or can burn the nerve. If significant damages are observed in the nerves, we recommend reducing the electrical pulse intensities or placing the electrode further away from the nerve.
In our present protocol, CAG promoter-driven plasmids were used as expression vectors. CAG promoter-driven plasmids allow high levels of gene expression in myelinating SCs in vivo. We also have tried a CMV promoter, another widely used universal promoter for mammalian gene expression, but expression of the gene product was very weak. This is consistent with previous results, in which electroporation-mediated transfection was conducted in the embryonic brain20. Therefore, we recommend using CAG promoter-driven plasmids for the in vivo electroporation method.
Because axonal signaling is a key factor in myelination/demyelination21, gene modification in neurons is also important. However, delivery of transgenes using our in vivo electroporation method is limited to SCs. It has been reported that gene delivery into sciatic nerve axons can be achieved when in vivo electroporation is applied to dorsal root ganglion (DRG) neurons in adult rats22. This suggests that delivery of plasmid DNA to the cell body is likely to be critical for in vivo transfection of peripheral axons. Thus, to examine the involvement of axonal molecules in myelination/demyelination, researchers should use neuron-specific genetic methods such as genetically modified animals, neuron-specific viral vectors, or in vivo electroporation to DRG neurons.
Compared with current methods, such as the generation of genetically modified animal lines23 and delivery of transgenes by viral vectors4-6, gene modification of SCs by in vivo electroporation is simpler. This method only requires several days for plasmid DNA construction and one day for electroporation surgery. Plasmid DNA construction does not require a biohazard room that is usually essential for viral vector handling. In addition, one of the advantages of the electroporation method is the capacity for simultaneous expression of multiple gene products using a simple protocol. Our novel technique will be useful for analyzing the interaction of a variety of signaling molecules involved in myelination and demyelination. In particular, by permitting the cotransfection of a number of different intracellular fluorescent probes, our method should be a powerful tool for investigating intracellular signaling dynamics in SCs using live imaging experiments.

The rapid transmission of sensory and motor information in the peripheral nervous system is permitted by the myelin sheath, which is formed by myelinating Schwann cells (SCs)1. Insulation of axons by the myelin sheath enables saltatory conduction, which increases the speed of nerve impulses. In disorders in which the development or maintenance of the myelin sheath is impaired, nerve conduction speed is reduced. This results in neuropathy involving motor and sensory dysfunction. Although there are many studies on the molecular mechanisms of myelination and demyelination in the peripheral nervous system, the roles of the numerous proteins involved in these processes remain unclear.
To study the molecular mechanisms of SC myelination/demyelination in vivo, genetic approaches have been used to modify gene expression in animals. A powerful approach is the use of knockout/knockin or transgenic animals. However, the generation of these animals is expensive and time consuming. For SC-specific gene manipulation, crossing floxed strains with Cre mice or other conditional gene expression methods are necessary. This again is laborious and time intensive. In recent years, a cutting-edge genetic technology, the CRISPR-Cas9 system, has made the generation of genetically modified mice much quicker (about 4 weeks)2,3, but this method is hindered by target sequence limitations, and suffers from off-target effects. As an alternative method, viral vector-mediated gene transfer is a faster and easier method of achieving gene transfer into SCs in vivo4-6. Indeed, the generation of viral vectors is less expensive, and takes a shorter time (within a few weeks), and gene manipulation of SCs can be achieved by simply injecting engineered viral vectors, such as adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors, into the sciatic nerve. Because these viral vectors have different characteristics, users have to choose the one best suited for their purpose. Adenoviral vectors infect axons and SCs in both young and mature sciatic nerves. In particular, adenoviral vectors have higher selectively for non-myelinating SCs than myelinating SCs. Adenoviruses can cause immune responses, and accordingly, immunodeficient strains should be used5. AAV vectors are currently the most widely used viral vectors, and allow in vivo gene transfer with lower toxicity7. AAV can transduce both axons and SCs by direct injection into the nerve fibers8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels7,9. Therefore, it is difficult to analyze myelination, which actively progresses during the two week postnatal period. Lentiviral vectors have higher selectively for myelinating SCs than non-myelinating SCs, and do not have toxic effects on sciatic nerves. However, lentiviral vectors do not infect SCs in more mature nerves5, and therefore are unsuitable for analyzing events such as the demyelination process.
Electroporation is another faster and easier approach to achieve in vivo gene transfer. It has been reported that in vivo transfection of SCs can be achieved when electroporation is applied to transected rat sciatic nerves10. However, because this method requires nerve transection for gene delivery, the application is limited to the analysis of the damaged nerves. Here, we describe an alternative method that allows the delivery of transgenes into myelinating SCs in intact rat sciatic nerves using electroporation11. This method requires plasmid construction, which can usually be completed within a week. Then, by simply delivering electric pulses to the site on the sciatic nerve where the plasmid DNA was injected, highly selective transfection of myelinating SCs can be achieved in neonatal as well as in more mature animals. By electroporating multiple plasmids, simultaneous expression of a variety of genes can be easily achieved. The ability to simultaneous express multiple molecules, such as signaling proteins, short-hairpin RNAs (shRNAs) and functional probes, is crucial for investigating complex processes such as myelination and demyelination. The novel in vivo electroporation method described in this paper will be a powerful tool, allowing researchers to analyze the function of a multitude of molecules and their interactions in myelinating SCs.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genopure Plasmid Maxi Kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;">03 143 422 001</td>
			<td style="width:167px;">Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green CFC</td>
			<td>WAKO</td>
			<td>069-00032</td>
			<td>Dye for DNA injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC 150T-10</td>
			<td>HARVARD APPARATUS</td>
			<td>30-0062</td>
			<td>Glass capillary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction tubing</td>
			<td>Drummond</td>
			<td>05-2000-00</td>
			<td>Suction tubing for micro injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MODEL P-97</td>
			<td colspan="2">SUTTER INSTRUMENT CO.</td>
			<td>Micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CUY21 Single Cell</td>
			<td>BEX</td>
			<td>Electroporator CUY21 Single Cell</td>
			<td>Pulse generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric warmer</td>
			<td>KODEN</td>
			<td style="width:119px;">CAH-6A</td>
			<td>Warmer during the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isofluolane</td>
			<td style="width:71px;">Mylan</td>
			<td style="width:119px;">1119701G1076</td>
			<td>Anesthetic</td>
		</tr>
	</tbody>
</table>

in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

プラスミド発現性赤色蛍光タンパク質（RFP）でトランスフェクトした坐骨神経の例は、図2Aに示されています。双極性形態を示す細胞は、SCの特徴は、まばらにRFPをトランスフェクトしました。いいえRFPの蛍光は軸索で検出されませんでした。我々は通常〜すべての神経で100トランスフェクトのSCを見つけます。このトランスフェクション効率は、レンチウイルスベクター4を用いたin vivo SC感染効率に似ているようです。 免疫染色実験は、ほとんど（〜96％）P7のS100、SCマーカー（ 図2B）のための共同標識し、P14のでRFP陽性細胞の91％MBPのための共同標識、髄鞘形成のSCマーカーでRFP陽性細胞（ 図2C）、エレクトロポレーションによる遺伝子導入は、SCのを髄鞘形成するための高度に選択的であることを示唆しています。 SCへの複数の遺伝子の導入<ein vivoでのmは&gt;髄鞘形成/脱髄のメカニズムを調査するために非常に有用であろう。ここに記載のin vivoエレクトロポレーション法の主な利点は、簡単な手順で複数の遺伝子を導入するための能力です。図2Dは​​、in vivoエレクトロポレーションに使用して、GFPおよびRFPを発現するプラスミドの混合物を用いてトランスフェクト坐骨神経の代表画像を示しています。 SCの約97％は、複数の遺伝子の非常に効率的な送達は、単に複数のプラスミドの混合物をエレクトロポレーションによって達成することができることを示唆し、二重陽性GFPおよびRFPました。 げっ歯類では、ミエリン形成が劇的生後最初の2週間の間に増加し、出産の周りに開始し、その後徐々に減少します。このように、遺伝的にこれらの発達の時間窓の間のSCを操作することによって、髄鞘形成のこれらの異なる段階のメカニズムを明確にすることができます。レンチウイルスベクターはのための良いツールです彼らは最小の毒性を持っている、特にとして、髄鞘形成をnalyzingが、レンチウイルスは唯一の新生児の坐骨神経の5,6に感染します 。トランスフェクションは（ 図2E、下）P3（ 図2E、上）またはP14に実施された場合と比較して、エレクトロポレーション媒介遺伝子導入はうまく動作します。 生体内エレクトロポレーション法における新規のアプリケーションがここで説明されている。 図3Aは、GFPを発現する種々の発生段階（P7、P14、P21とP31）で、ミエリン形成のSCの光学顕微鏡像を示します。光学顕微鏡分析により、そのような長さと直径との形態学的パラメータの変化を評価することができます。これらのパラメータは、エレクトロポ神経が重大な損傷を及ぼすことなく開発することを示唆し、無傷のラット末梢神経15,16と比較して同様の値を持っていることに注意してください。 図3Bは、LacZをexpreの電子顕微鏡像を示しますミエリン化のSCをssing。この場合には、LacZの発現をマーカーとして使用しました。 bluo-galを、エタノール不溶性基質を用いて、βガラクトシダーゼ染色、電子顕微鏡11,17によりトランスフェクトされたSCのミエリン構造の解析を可能にします。これらの実験では、シグナル伝達分子の役割は、それによって、機能喪失または機能獲得型の影響の分析を可能にする、それらの発現をサイレンシングするか、増強することによって調べることができます。固定組織の分析に加えて、 インビボでのエレクトロポレーション媒介遺伝子移入はまた、イメージング実験をライブに適用することができます。例えば、 図3Cは、G-GECO1.1 18、緑色蛍光細胞質ゾルのCa 2+インジケーター、およびR-GECO1mt 19、赤色蛍光ミトコンドリアのCa 2+指示薬を共発現ミエリンSCを示しています。これらの指標を発現することによって、我々はのSCのミエリン形成にサイトゾルおよびミトコンドリアのCa 2+濃度を制御するシグナル伝達経路を同定し<html> 。したがって、本発明の方法は、遺伝的にコードされた蛍光プローブは、関心のある信号を検出するために利用可能である場合は特に、シグナル伝達機構の種々の研究に使用することができます。 <img alt="図1" src="/files/ftp_upload/54567/54567fig1.jpg" /> 図 1：I Nインビボ 電気穿孔方法 の概略まず、麻酔したラットの坐骨神経を露出させます。第二に、プラスミドDNAは、坐骨神経に注入されます。第三に、電気パルスは、鉗子状の電極を介して注入部位に送達されます。最後に、傷は接着剤で閉じられています。この手順では、反対側の神経に繰り返すことができる。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54567/54567fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> &quot;&gt; <img alt="図2" src="/files/ftp_upload/54567/54567fig2.jpg" /> 図2： トランスフェクトされた坐骨神経上の代表的な結果 （A）トランスフェクトされた坐骨神経の代表画像。神経はP3でRFPを発現するプラスミドでトランスフェクトし、P7に固定しました。 （B）S100、SCマーカーとの共局在化を示すP7でのRFP-トランスフェクトされた細胞の代表画像。 MBP、髄鞘形成のSCマーカーと共局在化を示すP14でRFPトランスフェクト坐骨神経の（C）A代表画像。 （D）GFPおよびRFPを発現するプラスミドで同時トランスフェクション坐骨神経の代表画像。トランスフェクトされたSCが同時にGFPおよびRFPを発現しました。 （E）ミエリン形成が始まるP3、（上）でトランスフェクトP31でのSCをミエリン形成の画像、および最も大きな軸索 ​​は（下）有髄なっP14、でトランスフェクトP31におけるミエリン形成のSCの画像、そのTRANSFを示唆しています髄鞘形成のSCのectionは、新生児の神経ではなく、より成熟した神経だけでなく達成することができます。スケールバー=200μmの（A）。 50ミクロン（BE）。この数値は、当社の以前の出版物11から変更されました。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54567/54567fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54567/54567fig3.jpg" /> 図 3：in vivoでの エレクトロポレーション （A） のアプリケーションのSCをミエリン形成の発展の光顕微鏡分析。坐骨神経はP3でのGFP発現プラスミドをエレクトロポレーションした、と様々な発達段階（P7、P14、P21とP31）で固定しました。 GFP陽性SCの代表的な画像は、左側に示されています。平均長さと直径は、（n = 3の平均±SEMとしてまとめられています右側の3神経）47から0。開発が進むにつれてのSCが増加するミエリン形成の長さと直径。 （B）をコードするプラスミドのLacZでトランスフェクトされた坐骨神経の電子顕微鏡像。 （左白アスタリ​​スク）トランスフェクトされたSCが細かくβガラクトシダーゼ反応生成物の析出物で標識しました。 （C）G-GECO1.1、緑色蛍光細胞質ゾルのCa 2+インジケーター、およびR-GECO1mt、赤色蛍光ミトコンドリアのCa 2+指示薬で同時トランスフェクションSCの画像。白い点線の長方形内の領域は、右側のパネルに拡大して示されています。スケールバー=50μmの（AおよびC） 1ミクロン（B）。この数値は、当社の以前の出版物11から変更されました。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54567/54567fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

in-vivo-gene-transfer-to-schwann-cells-rodent-sciatic-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

8.8K ビュー.  The University of Tokyo. Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

ビデオ: In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

ラット坐骨神経からの軸索原形質分離

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

神経科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

開発マウス内耳への遺伝子導入による<emインビボで&gt;</em&gt;エレクトロポレーション

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

末梢神経損傷後の機能回復を評価する嗅神経鞘細胞の移植

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

マウス坐骨神経に生体内電気生理学的測定値の

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

インビボシングルファイバー記録と無傷の後部根神経を用いて伝導障害のメカニズムを調べる

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...