(F q 受賞資金が供給された研究Z.) NIH (R01NS064288、R01NS085176、R01GM111514、R01EY027347)、クレイグ H. Neilsen 財団、BrightFocus 財団によって。

ジョンズ ・ ホプキンス機関動物ケアおよび使用委員会によって承認された動物のプロトコルに従ってすべての動物実験を行った。
1. 材料および試薬
<ol><li>
動物
	<ol>
<li>実験のための 30-35 g の重量を量る 6 週齢雌 CF1 マウスを使用します。 注: マウスだったグループは建物 (5 マウス ケージごと) 1/4"トウモロコシ穂軸ベッド、2" 平方 nestlets ネスティングのために滅菌個別換気ケージの中で。ケージされた制御された 12 時間の明暗周期で維持され、部屋の温度は 19.4 ° C から 25 ° Cマウスは、グローバルの齧歯動物の食事と自動水供給システムで飼育しました。</li>
	</ol>
</li>
	<li>
楽器
	<ol>
<li>次の手術器具を使用して、効率的に手術を実施する: 腹腔内注射用使い捨て注射器 (27 G、1.0 mL) 毛皮のクリッパー、ステレオ解剖顕微鏡、マイクロ手術鉗子、マイクロ外科はさみ、小さな骨 rongeurs滅菌不織スポンジ。 注: すべての機器、材料、手術前にオートクレーブまたは生産者によって殺菌済み。手術中に楽器は滅菌を維持するために 75% アルコールを吹き付けています。</li>
	</ol>
</li>
	<li>
麻酔のソリューション
	<ol>
<li>麻酔を誘導し、DRG 注入前に麻酔解決策 #2 を使用して麻酔を維持するために麻酔液 #1 を使用してください。<ol>
<li>麻酔液 #1 をするためには、ケタミン ・ キシラジン最終濃度 10 mg/mL と 1.2 mg/mL に滅菌生理食塩水で希釈します。</li>
			<li>麻酔解決策 #2 をするためには、タヴェルタン最終濃度 20 mg/mL に滅菌生理食塩水で希釈します。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
プラスミド DNA と RNA Oligos
	<ol>
<li>次の製造元のプロトコルに応じて市販のキットを使用してプラスミッドを準備します。プラスミド Dna 濃度 2.0 μ g/μ L の滅菌の脱イオン水で希釈します。</li>
		<li>0.005-0.01 の最終濃度に到達する高速グリーン色素溶液を追加注入中に DRG アウトラインの視覚化に適した % (v/v)。</li>
		<li>50 μ M の最終濃度に到達するメーカーから提供される対応するバッファーでマイクロ Rna、siRNA または oligos を溶解します。</li>
	</ol>
</li>
	<li>
マイクロインジェクション ピペット
	<ol>
<li>マイクロ ピペットの引き手を使用して毛細管ガラスを引き、おおよそ 50 μ m 外径の開口部を生成するマイクロサージェリー ハサミで引っ張られた毛細管ガラス ピペットの先端をカットします。その後、約 20-30 分のためクリーン ベンチでの紫外線の下でガラス ピペットを滅菌します。</li>
		<li>細胞内のマイクロインジェクションに接続して滅菌ガラス ピペット ピペット ホルダーのマウントは調剤システムです。30 psi の圧力の期間の 8 ms でのインジェクション システムのパラメーターを設定します。</li>
	</ol>
</li>
	<li>
エレクトロポレーション システム
	<ol>
<li>方形波エレクトロポレーション システムに電極を接続し、次のパラメーターを設定:生体内でエレクトロポレーション用 950 ms 間隔で 35 V で 15 ms パルス。</li>
	</ol>
</li>
	<li>
血流とソリューションを修正
	<ol>
<li>4% (w/v) の濃度で 1 × PBS のパラホルムアルデヒド (PFA) 粉溶解します。4 ° C で PFA ソリューションを格納します。</li>
	</ol>
</li>
</ol>2 実験手順
<ol><li>
L4 と L5 Drg の手術手技
	<ol>
<li>#1 以前 (とケタミン 100 mg/kg 体重とキシラジン 12 mg/kg 体重) 準備麻酔液の腹腔内注入を使用するマウスを麻酔します。</li>
		<li>毛皮クリッパーで外科領域を剃る。 メモ: 左の坐骨神経のクラッシュのための別の手術がある、ので左大腿後部の毛皮が剃毛同時に。</li>
		<li>温水毛布 (35.0 ° C) の上にマウスを置くし、温度プローブで直腸の温度を監視します。</li>
		<li>麻酔をテストするには、つま先とマウスの尾をピンチ、行動反応を観察します。</li>
		<li>コルクボードの上でマウスの 4 つの手足をテープします。</li>
		<li>皮膚を切断する前に、iodophor を削除する 75% アルコール iodophor ソリューションとし、外科領域を拭いてください。</li>
		<li>麻酔中の乾燥を防ぐため目に眼軟膏を適用します。</li>
		<li>切開する前に罰金のマーカーペンと腸骨稜の両側をマークします。L5 Drg の位置の識別を容易にする腸骨稜の 2 つの点を結ぶ直線を描画します。</li>
		<li>マイクロ剪刀と腰痛の正中線に沿って 3 cm の切開を行います。また、毛様体筋 multiﬁdus と毛様体筋最長筋腰、S1 棘突起に L3 からなど、棘筋を外し、5 L4 と L5 6 椎間関節を公開します。</li>
		<li>5 L4 と L5 6 椎間関節を削除するのにには、マイクロ rongeur を使用します。また、L4、L5、Drg の背側を公開する左の神経弓を削除します。</li>
	</ol>
</li>
	<li>
DRG 注入
	<ol>
<li>(200 mg/kg 体重をタヴェルタン) と麻酔解決策 #2 を挿入腹腔 DRG 注射の前に麻酔を維持するために。</li>
		<li>ガラス毛細管ピペットにプラスミド DNA または RNA oligos (DRG あたり 1 μ L) を読み込みます。</li>
		<li>DRG に毛細管ガラス ピペットの先端を慎重に挿入します。</li>
		<li>DNA プラスミドの 1.0 μ L 溶液を注入して徐々 にまたはに後根神経節細胞内マイクロインジェクションを使用して RNA oligos 塗布システム (30 psi、8 ms)。 注: 注入の期間は、5 分を持続させるべき。</li>
	</ol>
</li>
	<li>
エレクトロポレーション
	<ol>
<li>電極の先端に PBS をドロップします。</li>
		<li>正方形の滅菌ガーゼで出血をクリーンアップします。</li>
		<li>電極とターゲット DRG を軽くピンチ、エレクトロポレーション システム 5 正方形電気パルスを適用します。</li>
		<li>それぞれ 5-0 ナイロン縫合糸で筋肉と皮膚の層を閉じます。</li>
		<li>それは完全に胸骨の横臥を維持するために十分な意識を取り戻したまで細心の注意の下で温水毛布 (35.0 ° C) の上にマウスを配置します。麻酔から完全に回復した後マウスを家のケージに戻ります。 注: それは 37 ° C 毛布に麻酔から完全に回復するためにマウスの約 60 分かかります。手術後の痛みを和らげるために給餌の 1 つ (200 mg) の丸薬イブプロフェンを 1,000 ml の水 (0.2 mg/ml) に溶解します。</li>
	</ol>
</li>
	<li>
坐骨神経のクラッシュ
	<ol>
<li>DRG エレクトロポレーション後 (実験計画) によって 2 つまたは 3 日間は麻酔麻酔液 #2 (400 mg/kg 体重をタヴェルタン) と腹腔内でマウスです。</li>
		<li>コルクボードの上でマウスの 4 つの手足をテープします。</li>
		<li>正中線に沿って左側に 1 cm の切開 0.5 cm を作る。大殿筋や梨状筋などの筋肉を縦にカットします。大坐骨孔と坐骨切痕の間坐骨神経の部分を公開します。</li>
		<li>12 s のマーク 10-0 ナイロン epineural 縫合糸でクラッシュ サイト手術鉗子で神経のクラッシュ。クラッシュ サイトをマークする硬膜に結び目を作る。</li>
		<li>5-0 ナイロン縫合糸で筋肉と皮膚の層を閉じます。</li>
		<li>それは完全に胸骨の横臥を維持するために十分な意識を取り戻したまで細心の注意と温水毛布 (35.0 ° C) の上にマウスを配置します。麻酔から完全に回復した後マウスを家のケージに戻ります。 注: それは 37 ° C 毛布に麻酔から完全に回復するためにマウスの約 60 分かかります。手術後の痛みを和らげるために給餌の 1 つ (200 mg) の丸薬イブプロフェンを 1,000 ml の水 (0.2 mg/ml) に溶解します。</li>
	</ol>
</li>
	<li>
マウス灌流、DRG、坐骨神経の収穫
	<ol>
<li>2 つまたは 3 日間 (実験計画) によって坐骨神経のクラッシュ後のマウス腹腔内麻酔解決策 #2 を麻酔します。</li>
		<li>冷えた 4% パラホルムアルデヒド (PFA) (pH 7.5) PBS で続く PBS (pH 7.4) とマウス transcardially を灌流します。</li>
		<li>後、マイクロ剪刀と解剖顕微鏡マイクロ鉗子一緒に神経根や坐骨神経膝に Drg を慎重に区切ります。4 %pfa 一晩 4 ° C で坐骨神経を直接に</li>
	</ol>
</li>
	<li>
イメージングと蛍光標識感覚軸索を測定
	<ol>
<li>接続されている組織、マイクロ剪刀と解剖顕微鏡の下で慎重にマイクロ鉗子で固定の坐骨神経の膜をはがします。4% を交換し、十分に洗うと PBS PFA 神経 3 回します。</li>
		<li>スライドの坐骨神経を置き、それをまっすぐに保ちます。Antifade ソリューション、神経の周りの 80 μ L を追加し、それを観察を置きます。圧力マウント全体の組織をフラット化します。</li>
		<li>モザイク集録および画像処理用アクセサリーを搭載した倒立 epiﬂuorescent 顕微鏡に ﬂattened 組織を配置します。 注: 励起と放射距離は、488 nm および 509 nm。また、手動で複数の重複した画像を取るし、一緒にプラグインのモザイクで ImageJ を用いた画像をステッチすることが可能です。</li>
		<li>再生軸索の長さを測定する場合は、すべて identiﬁable ﬂuorescently ラベルにおける軸索遠位軸索の端に (10-0 epineural 縫合でマーク) クラッシュ サイトから坐骨神経をトレースします。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Saijilafu, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K-GSK3+signalling+regulates+mammalian+axon+regeneration+by+inducing+the+expression+of+Smad1.">PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1.</a> Nature Communications. 4, 2690 (2013).</li><li>Zhang, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt-independent+GSK3+inactivation+downstream+of+PI3K+signaling+regulates+mammalian+axon+regeneration.">Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration.</a> Biochemical and Biophysical Research Communications. 443 (2), 743-748 (2014).</li><li>Jiang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+supports+mammalian+axon+regeneration+in+vivo+by+suppressing+GSK3beta+expression.">MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression.</a> Cell Death &amp; Disease. 6, 1865 (2015).</li><li>Krames, E. 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著者が明らかに何もありません。

いくつかの手術の手順には、特に注意が必要です。L4 と L5 drg コード (細胞体の位置) は、坐骨神経を支配を正しく識別され、遺伝子構造に注入する必要があります。そうでなければ、GFP 標識が休演いたします坐骨神経軸索で。腸骨稜は、L4 と L5 Drg を特定する有用な解剖学的ランドマークとして見なすことができます。L5 と L6 の椎間関節面は、ほとんどマウス腸骨稜12に近?...

神経外傷による神経系の傷害または様々 な神経変性疾患は、通常、モーター、感覚と認知機能の欠陥になります。最近では、多くの努力は、負傷した生理機能ニューロン1,2,3を復元する大人ニューロンの再生力再建に専念してきました。DRG ニューロンは痛み、温度、タッチ、または脳への姿勢など、さまざまな感覚刺激を伝達する神経細胞のクラスターです。各これらのニューロンは擬似単極、周囲に向かって拡張 1 つの支店と4脊髄に向かって他の支店を分割単一の軸索が含まれています。Drg の大人の感覚ニューロンはいくつか成熟した哺乳類神経損傷後の軸索を積極的に再生する知られています。したがって、感覚軸索の損傷広範囲として採用されている重要なモデル軸索再生の生体内のメカニズムを研究します。
生体内で遺伝子トランスフェクション技術は、通常以下をセットアップする時間がかかり、トランスジェニック動物を使用するよりもより柔軟な遺伝子の機能を勉強し、中枢神経系における情報伝達システムに必須の役割を果たしています。主な技術は、2 つの方法に分類できます: 計測器およびウイルス ベース5。大人ニューロンの遺伝子デリバリー ウイルス ベースは生体内で遺伝子発現6の正確な時空間操作を提供できます。しかし、労働集約的なプロセスは生産目的の遺伝子を含んでいるウイルス粒子の精製など、ウイルス ベースの方法で関与しています。さらに、多くのウイルスのベクトルは、データ集録、データの解析を妨げる可能性があり、可能性の実験結果の解釈を誤解するホストの免疫システムをアクティブにでした。エレクトロポレーション、典型的な楽器ベースのトランスフェクション アプローチ遺伝子ベクターまたは7 細胞外スペースから小さな RNA oligos の流入を支持の一過性細胞および原子膜の透過性を高める電気パルスを使用して、.体外エレクトロポレーションは、さまざまなタイプのターゲット遺伝子発現を操作するための一時的な非常に効率的な戦略として認識されています。体内エレクトロポレーションは、効率が低い株ウイルスのベクトルと比較しての一時的な遺伝子発現に他ならない、ウイルスのアプローチに比べて様々 な利点があります。たとえば、ほとんどすべて組織・細胞7,8,9に適用できます。さらに、特定の成績に対してどちらかのプラスミド (例えばSirna、マイクロ Rna) の興味の遺伝子または小さな RNA oligos 標的組織に直接注入でき、電気的パルス、手順にして以下の労働 - と時間のかかります。また、複数のプラスミドと単一エレクトロポレーションと同時に RNA oligos の株は、可能です。
我々 が成体マウス感覚神経細胞における遺伝子発現を操作する生体内でエレクトロポレーション アプローチを確立が正常に適用され、1,パイオニア研究多数2,3 でこのようなアプローチを検証 ,8,10。ここでは、哺乳類の軸索の再生の将来の研究のためのこのアプローチの使用を容易にするために詳細なプロトコルを提案する.

<table><tbody><tr><td>ECM 830 Square wave electroporation system</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>For &lt;em&gt;in vivo&lt;/em&gt; electroporation</td></tr><tr><td>Tweezertrodes electrodes</td><td>BTX Harvard Apparatus</td><td>45-0524</td><td>For &lt;em&gt;in vivo&lt;/em&gt; electroporation, 1 mm flat</td></tr><tr><td>Picospritzer III</td><td>Parker Instrumentation</td><td>1096</td><td>Intracellular Microinjection Dispense Systems</td></tr><tr><td>Glass Capillary Puller</td><td>NARISHIGE</td><td>PC-10</td><td></td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>1902328</td><td></td></tr><tr><td>Stereo Dissection Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Microsurgery Rongeur</td><td>F.S.T</td><td>16221-14</td><td></td></tr><tr><td>Microsurgery Forceps</td><td>FST by DUMONT, Switzerland</td><td>11255-20</td><td>Only for sciatic nerve crush</td></tr><tr><td>Glass Capillary</td><td>World Precision Instruments, Inc.</td><td>TW100-4</td><td>10 cm, standard wall</td></tr><tr><td>Tape</td><td>Fisherbrand</td><td>15-901-30</td><td>For fixing the mouse on the corkboard</td></tr><tr><td>2, 2, 2-Tribromoethanol (Avertin)</td><td>Sigma-Aldrich</td><td>T48402</td><td>Avertin stock solution</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>152463</td><td>Avertin stock solution</td></tr><tr><td>siRNA Fluorescent Universal Negative Control #1</td><td>Sigma-Aldrich</td><td>SIC003</td><td>Non-target siRNA with fluorescence</td></tr><tr><td>microRNA Mimic Transfection Control with Dy547</td><td>Dharmacon</td><td>CP-004500-01-05</td><td>Non-target microRNA with fluorescence</td></tr><tr><td>Plasmids preparation kit</td><td>Invitrogen Purelink</td><td>K210016</td><td>GFP-coding plasmid preparation</td></tr><tr><td>Fast Green Dye</td><td>Millipore-Sigma</td><td>F7252</td><td>For better visualization of the DRG outline during injection</td></tr><tr><td>Ketamine</td><td>Putney, Inc</td><td>NDC 26637-731-51</td><td>Anesthesia induction</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td>NDC 59399-110-20</td><td>Anesthesia induction</td></tr><tr><td>Acetaminophen</td><td>McNeil Consumer Healthcare</td><td>NDC 50580-449-36</td><td>Post-surgical pain relief</td></tr></tbody></table>

investigating mammalian axon regeneration: in vivo electroporation of adult mouse dorsal root ganglion

エレクトロポレーションは、DNA プラスミッドまたは小さな RNA 分子を細胞に導入する本質的な非ウイルス性遺伝子導入アプローチです。後根神経節 (Drg) の感覚ニューロンは、2 つの分岐の 1 つの軸索を拡張します。1 つの分岐は、周辺機器に行く神経 (末梢枝) と他の支店 (中央支部) 後根を介して脊髄に入る。神経損傷後末梢枝は中央支店、再生成しないに対し力強く再生されます。ため高い再生能力感覚軸索の再生は広く使用されてモデル システムとして末梢神経系 (PNS)、中枢神経系 (CNS) の両方で哺乳類の軸索の再生を研究します。ここでは、成熟したニューロンの生体内でエレクトロポレーションによる遺伝子発現を操作する以前に確立されたアプローチ プロトコルについて述べる。アプローチ芳司プラスミッドまたは小さな RNA oligos (Sirna またはマイクロ Rna) に基づき、遺伝子の利益または軸索再生の生体内の調節におけるマイクロ Rna の役割を検討する両方の損失と利得-の機能実験のためことができます。さらに、空間的そして一時的は比較的短時間で遺伝子発現の体内操作の制御ができます。このモデル系は、哺乳類の軸索の再生が規制生体内では分子メカニズム解明のためのユニークなツールを提供します。

L4 と L5 Drg に DRG エレクトロポレーション体内のトランスフェクション率は十分に高く、注入我々 を検証し、現在のプロトコルの細胞毒性や electroporated 蛍光タグ付きマイクロ Rna、siRNA を定量化します。戸建の Drg を用いて電子顕微鏡や免疫組織化学 (図 1A B) 経由で処理されました。注射エレクトロポレーション後の細胞生存?...

Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

エレクトロポレーションは、興味の遺伝子を細胞に提供する効果的なアプローチです。このアプローチは生体内で成体マウス後根神経節 (DRG) のニューロンに適用すると、軸索再生の生体を研究するためのモデルについて述べる。

成体マウス後根神経節の調査の哺乳類の軸索の再生: In Vivoエレクトロポレーション

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

investigating-mammalian-axon-regeneration-vivo-electroporation-adult

Research

JoVE Journal

Neuroscience

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

8.2K ビュー.  Johns Hopkins University School of Medicine. エレクトロポレーションは、興味の遺伝子を細胞に提供する効果的なアプローチです。このアプローチは生体内で成体マウス後根神経節 (DRG) のニューロンに適用すると、軸索再生の生体を研究するためのモデルについて述べる。

ビデオ: Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

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 ...

神経科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

軸索損傷後の脊髄後根神経節の組織からのクロマチン免疫沈降

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

根切断術後の後根の軸索のライブイメージング

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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.

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 ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

後根神経節インジェクションおよび後根感覚軸索再生のためのモデルとして傷害をつぶします

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

アキソトマイズ化ラットレチナル神経節ニューロンの嗅覚エンシアシンググリアの共培養、成人軸索再生のインビトロモデルとしての

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

ラット背根神経節外植片とシュワン細胞の共培養における末梢軸索のin vitro髄鞘形成

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

イムノパンニングによる成体マウス背根神経節培養の濃縮

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...