(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 border="0" cellpadding="0" cellspacing="0" style="width:1039px;" width="1037">
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		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">ECM 830 Square wave electroporation system</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0052</td>
			<td style="width:351px;">For in vivo electroporation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">Tweezertrodes electrodes</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0524</td>
			<td>For in vivo electroporation, 1 mm flat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Picospritzer III</td>
			<td style="width:232px;">Parker Instrumentation</td>
			<td style="width:121px;">1096</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary Puller</td>
			<td style="width:232px;">NARISHIGE</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:121px;">1902328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stereo Dissection Microscope</td>
			<td style="width:232px;">Leica</td>
			<td style="width:121px;">M80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Rongeur</td>
			<td style="width:232px;">F.S.T</td>
			<td style="width:121px;">16221-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Forceps</td>
			<td style="width:232px;">FST by DUMONT, Switzerland</td>
			<td style="width:121px;">11255-20</td>
			<td>Only for sciatic nerve crush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary</td>
			<td style="width:232px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW100-4</td>
			<td>10 cm, standard wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tape</td>
			<td style="width:232px;">Fisherbrand</td>
			<td style="width:121px;">15-901-30</td>
			<td>For fixing the mouse on the corkboard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2, 2, 2-Tribromoethanol (Avertin)</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">T48402</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-2-butanol</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">152463</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">siRNA Fluorescent Universal Negative Control #1</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">SIC003</td>
			<td>Non-target siRNA with fluorescence</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:335px;">microRNA Mimic Transfection Control with Dy547</td>
			<td style="width:232px;">Dharmacon</td>
			<td style="width:121px;">CP-004500-01-05</td>
			<td>Non-target microRNA with fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmids preparation kit</td>
			<td style="width:232px;">Invitrogen Purelink</td>
			<td style="width:121px;">K210016</td>
			<td>GFP-coding plasmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fast Green Dye</td>
			<td style="width:232px;">Millipore-Sigma</td>
			<td style="width:121px;">F7252</td>
			<td>For better visualization of the DRG outline during injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Ketamine</td>
			<td style="width:232px;">Putney, Inc</td>
			<td style="width:121px;">NDC 26637-731-51</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Xylazine</td>
			<td style="width:232px;">AnaSed</td>
			<td style="width:121px;">NDC 59399-110-20</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Acetaminophen</td>
			<td style="width:232px;">McNeil Consumer Healthcare</td>
			<td style="width:121px;">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

8.1K 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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

後根潜在的記録によりマウスにおける脊髄シナプス前抑制を測定する<em&gt;インビボ</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

後根神経節モデル：癌性神経浸潤のin vitroモデルでは

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

後根神経節ニューロンのアライメントや髄鞘形成を強化するためのアプローチ

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

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 ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

腰部後根神経節のアクセスと豚注入の椎

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

単一の坐骨神経とフローサイトメトリーを使用して単一のマウスの後根神経節における免疫系細胞の解析

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

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 ...