この作品は、クリストファーとデイナ・リーブ財団、医学研究評議会、欧州研究評議会ECMneuro、ケンブリッジNHMRC生物医学研究センターからの助成金によってサポートされていました。私たちは、撮影時の彼らの技術支援のためのヘリーン・メレルバン「トンSpijkerとユスティナバラットに心から感謝の意を表したいと思います。私たちは、AAVの生産を支援するために博士エリザベス・モロニー教授ジョースト・バーハーゲン（神経科学のためのオランダ研究所）に感謝したいと思います。

以下のすべての動物の手順はイギリスの動物に従って行った（科学的処置）法1986これらの手続きに慣れていない場合は、全国/地域の規制にチェックして、プロトコルを開始する前に、獣医の助言を求めてください。 1.動物の適切ひずみを選びます注：感覚と足求心路遮断の損失の後根挫滅損傷の結果。前足の求心路遮断の一般的な副作用は、オーバーグルーミング、自傷、および足の自切を含めることができます。 <ol><li>手続きのために動物を取得します。 注：ラットの場合、そのようなリスター・フードおよびWistar系などのより活性な株は、このようなルイスとのSprague-Dawley系のような、より従順株と比較して、求心路遮断後の足自切の発生率が高いです。可能であれば、最も従順な歪み、ルイスは、常に最初に考慮しなければなりません。特に活性株は、遺伝mまでの実験のために必要でなければなりませんodificationまたは特定の行動評価の要件は、獣医学的治療が期待される悪影響に対処するための場所である必要があります。 </li></ol> 2.注射用ウイルスの準備注意：生物学的および実験室安全規定に従ってすべてのウイルスを処理します。 <ol><li>個々のウイルスパッケージングプロトコル9以下、2×10 12ゲノムコピー（GC）/ mLの力価にウイルスを希釈または濃縮します。 注記：ここでは、ウイルスは、5％のスクロースをダルベッコのリン酸緩衝生理食塩水（D-PBS）で希釈しました。力価は、ウイルスの種類に応じて最適化する必要がある、プロモーター、サイズを構築、および細胞型を標的にします。 </li><li>オプション：後で注入を簡単に可視化のためのウイルス液に1％着色染料（ 例えば、ファストグリーン）を追加します。非常に優しくソリューションを混ぜます。 </li><li> （4時間以内）すぐに使用氷上で溶液を維持または長期のために-80℃の冷凍庫で長期保存。 </li></ol> 3.動物の術前の準備を行います注：手術の極端な侵襲性のため、無菌技術を常に使用する必要があります。 <ol><li>手術を開始する前に、すべての手術器具および脊髄定位フレームを準備し、滅菌します。例えば、134℃のオートクレーブ中でツールを殺菌します。各動物のための滅菌のツールの新鮮なセットを使用してください。 </li><li>動物を麻酔。引っ込め反射またはまぶたの反射のために角膜に触れることによって、テストするために足をつまんで、適切な麻酔を確認してください。 <ol><li>例えば、イソフルランなどの吸入麻酔を用いて、成体ラットについて、2.0 Lの4％イソフルランを使用/誘導のための酸素の分。過剰または過少投与を避けるために、動物の呼吸パターンに従ってイソフルラン濃度を変える、1.5 L /酸素の分3％イソフルラン - 手術全体メンテナンスのため、2を使用します。 </li></ol></li><li>に動物が最初に麻酔し、CE、動物の術前体重を記録します。 </li><li>子宮頸部DRG注入のため、肩甲骨のすぐ遠位の耳の間から首に毛を剃り落とします。防腐剤製品と剃らエリアを消毒します。 </li><li>適切な鎮痛剤の投与量（ 例えば、5ミリグラム/ kgのカルプロフェン）および抗生物質（ 例えば、5ミリグラム/ kgのエンロフロキサシン）とともに、動物の脇腹に生理食塩水を皮下の2ミリリットルを注入します。 </li><li>手続きの際に角膜の乾燥を防ぐために、両眼に眼軟膏を適用します。 </li><li>動物の下に配置された37℃での加熱パッド付き脊椎定位フレーム上の手術に進みます。 </li></ol> 4. DRGを注入し、後根のクラッシュ傷害の実行注：これは非常に繊細な手術です。動物の手術を生きることを進める前に、解剖学で理解するために、最初の数死んだ動物で練習することをお勧めします。 <ol><li> L肌の上に著名なC2およびT2棘突起をocate。皮膚が開かれると、白色の繊維状組織の中線は、筋肉の最初の層の上に表示されなければならない（10番のメスの刃でC2およびT2棘突起の間に皮膚切開を行い、筋肉の最初の層は、「有しますゼリー状」テクスチャ）。 </li><li>手術用メスの刃を用いた白色正中線に沿って筋肉の1層目と同様のサイズの切開を行います。そこの近くにある下行大動脈からの主要幹線の分岐があるとして、著名なT2棘突起を越えないでください。 注：重い出血以降、このステップからどこでも発生する可能性があります。必ず出血を停止し、すべての回で明確な可視化を可能にするために滅菌綿棒または外科吸収性スポンジを使用して血液を除去。手続きを進める「盲目的」後に動物に予期せぬ悪影響につながる、脊髄、DRG、または後根への不要な損傷をもたらすことができます。 </li><li>撤回2つの開創器、吻側に配置され1つずつ尾を使用して、筋肉の最初の層と筋の第2の層が、横紋外観と、表示されなければなりません。 </li><li> （二縦筋が薄く、膜状組織によって接続観察することができる）筋の第2の層の正中線を見つけます。二つの長手方向の筋肉を分離するmicroscissorsのペアを使用して膜状組織を切開。これは、不必要な筋肉の損傷や出血をもたらすことができるよう、可能な場合は手術用メスの刃を使用しないでください。 </li><li>脊椎を覆う薄い筋の第三の層を露出するために応じてリトラクターを調整します。棘突起を軽く筋の第三の層の上にピンセットで触れることによって感じることができます。 </li><li> microscissorsを使用して、筋肉の三層の上に小さな切開を行い、ゆっくりとはっきりと椎骨を露出するように横向き的にキューレットまたはメスを用いて骨から筋肉を削り取ります。必要な場合は、ラミを作るために離れて筋肉や腱の一部をカットnectomy容易になります。 </li><li>注意深く細かい骨鉗子のペアを使用して薄層と茎の一部を除去することにより、T1の椎骨 - 左C5を露出させる - C8のDRGを、C4の左半椎弓切除術を行います。 DRGは、椎骨の横孔の近くに位置しています。 注：C3 - C7椎骨が目立つ棘突起を持っていません。 C8 DRGはC7とT1椎骨の間であるC5 DRGは、C6 DRGのようにC5とC6の椎骨の間にある、そして、C4及びC5椎骨の間に配置されています。何C8の椎骨はC8 DRGの存在とC8脊髄セグメントにもかかわらず、ありません。 </li><li> DRGの十分な一回注入のために露出されている、定位シリンジホルダーにカスタムメイド、33ゲージ鈍針を取り付けたウイルスを充填したマイクロシリンジを配置することによって注射器を準備します。注射の前に、注射針の挿入を支援することを目的とDRGの各上の小さな表面的な開口部を作るために30ゲージベベル針を使用しています。 </li><li> 33-Gを挿入定位座標を調整するために穏やかにノブを回すことにより、DRGの中心部へのAuGe針。これは、流体がDRGの腹側から漏れるする原因となり、針を介して、挿入しないでください。 、発生漏洩すぐに針の位置を調整する必要があります。 注：数値の定位座標が使用されていません。しかし、注射用の針を保持するためのフレームを利用すると便利です。 </li><li>注入シリンジポンプを用いて0.2μL/分で各DRGへのウイルスの1μLを注入します。針を引き抜く前に、追加の3分間待ってください。ウイルス溶液が着色染料が含まれている場合は、注射時には、DRGはゆっくりと色が変わります。 </li><li>同時C5実行する - C8の後根圧潰損傷を、10秒間3回各完全鉗子の両端、先の細いピンセット（ボンマイクロ）のペアを使用して、各ルートをつぶします。組織内の白い線がクラッシュサイトで表示されます。 FORCで必要とされるよりも深く行ってはいけませんこのようEPSは、前根への損傷を引き起こす可能性があります。 </li><li>注入および/またはクラッシュの後、出血や動物を閉じる前に切開部位に残さ骨片の小片がないことを確認してください。好適な場合には、暴露脊髄およびDRGの上に手術用吸収性スポンジの小片を配置します。 </li><li>筋肉の第3層は縫合せずに脊椎に自然に戻って撤回することを許可します。緩く吸収6-0縫合材料と（≈3は、縫合糸を中断）は、第2の層の2つの長手方向の筋肉を縫合。筋肉の最初の層を縫合吸収6-0縫合材料と（≈5は、縫合糸を中断）。 </li><li>吸収5-0縫合材料（≈10縫合糸を中断）で皮膚を縫合。 注：縫合糸はあまりにもタイトではないことを確認してください。皮膚の膨らみは、過剰引き締めの兆候であると動物に不快感を引き起こす可能性があります。 </li><li>重い出血が手術中に発生した場合、皮下注射1 - 2地域の規制の下で許可された動物からの流体の損失を補充するための生理食塩水のミリリットル。 </li><li>食用水和ゲルを提供し、動物が麻酔から完全に回復することができ、バック保持領域に動物を戻す前に少なくとも1時間、定期的なモニタリングを行います。 </li><li>感覚軸索再生を評価するために、最適な導入遺伝子発現のために、少なくとも3週間は動物を保管してください。 </li></ol>動物の術後ケアを行う5. <ol><li>最初の一週間後に手術中の動物用食品のマッシュと柔らかい綿の寝具を提供します。必要であれば、全国/地域の規制や獣医の助言に従い、回復を支援するために鎮痛剤や抗生物質の追加的な用量を投与。 </li><li> 10日 - 7後に皮膚に縫合糸を外します。 注：手術の一般的な副作用は、切開部位に起因するかゆみのために皮膚に傷マークを血清腫や血腫の形成、内部吸収性縫合糸、微妙な感覚や運動不足、および後根挫滅損傷後の求心路遮断足に自傷の兆候。 、実質的に予想以上に深刻である任意の福祉の問題については、すぐに獣医の助言を求めます。 </li></ol> 6.軸索トレース用順行CTBの噴射を行います注：前の組織コレクションに週を追跡コレラ毒素Bサブユニット（CTB）軸索を実行することをお勧めします。 <ol><li>メーカーの指示に従って1％CTB溶液を調製します。 </li><li>オプション：後で注入を簡単に可視化するためのソリューションに1％着色染料を追加します。非常に優しくソリューションを混ぜます。 </li><li>動物を麻酔（ステップ3.2.1を参照）、テーブルに手足をテーピングすることにより、左前足を安定化させます。 </li><li>手動でゆっくりと皮下無毛フットパッドの中心に1％CTBの1μLを注入し、4桁の数字は、マイクロリットルシリンジ嵌合のWiを用いてカスタムメイド、33ゲージの短い針番目。 注：注射の前に、まず注射針の挿入を支援し、皮膚に小さな表面的な開口部を作るために30ゲージベベル針を使用することができます。 </li><li>動物が保持領域に動物を返す前に麻酔から完全に回復することを許可します。 </li></ol> 7.組織の収集<ol><li>免疫組織化学のためにウイルス注入DRGおよび脊髄を収集するために、麻酔薬の過剰投与を投与し、経冷4％パラホルムアルデヒドに続くリン酸緩衝生理食塩水で動物を灌流。慎重に、顕微鏡下で固定された組織を収集するために、背にフル椎弓切除術を行います。分析4、5、6の必要に応じて組織調製、切片、および処理を進めます。 注：Sの存在によって観察として回収切開部位は、明らかであるべきです車の組織。 </li><li>あるいは、in vitro培養のためにウイルス注入DRGを収集するために、そのような二酸化炭素の上昇する濃度として承認された方法を用いて人道的に動物を犠牲に。慎重に可能な限り無菌状態を確保すること、顕微鏡下でDRGを解剖。 4、5、6、10を所望のように組織培養を進めます。 </li></ol>

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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=Directed+evolution+of+a+novel+adeno-associated+virus+(AAV)+vector+that+crosses+the+seizure-compromised+blood-brain+barrier+(BBB).">Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).</a> Mol Ther. 18 (3), 570-578 (2010).</li><li>Fagoe, N. D., Attwell, C. L., Kouwenhoven, D., Verhaagen, J., Mason, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ATF3+or+the+combination+of+ATF3,+c-Jun,+STAT3+and+Smad1+promotes+regeneration+of+the+central+axon+branch+of+sensory+neurons+but+without+synergistic+effects.">Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects.</a> Hum Mol Genet. 24 (23), 6788-6800 (2015).</li></ol>

James Fawcett is a paid consultant for Acorda Therapeutics and Novartis.

この記事では、我々は、成体ラットの下頸髄におけるDRG注射および後根挫滅損傷を実行するためのステップバイステップのガイドを提示します。これは非常に侵襲的で繊細な手術であるように、私たちは強く、すべての潜在的なユーザーは、動物の手術を生きるために進む前に、十分な訓練と実践を取得することをお勧めします。ユーザーは、脊髄の解剖学ではなく、周囲の筋肉組織、椎骨?...

神経系損傷後の軸索再生を達成することは困難な作業である1。中枢神経系（CNS）における軸索再生の失敗を研究するために、研究者は神経損傷モデルの茄多を使用していました。 CNSの領域が異なるので、軸索再生を研究するために解剖学的に適切なモデルを使用することが重要です。適切なモデルを使用することにより、研究者は、「1対すべて」治療戦略とは反対に、傷害の重症度、関心の神経細胞の種類に基づいて特定の治療、および再生を評価するための必要な脊髄路を策定することができます。 脊髄損傷では、例えば、最も衰弱症状は感覚と運動の損失から生じます。運動の損失が下降運動経路への損傷によって引き起こされている間の感覚の喪失は、昇順感覚経路への損傷によって引き起こされます。これらTWの間の細胞および解剖学的な違いによるO経路、多くの標的に軸索再生の研究では、いずれか一方のみの成功回復が患者に多大な利益となることを根拠に、どちらか一方の経路に焦点を当てます。この記事では、ウイルスベクターおよび感覚軸索再生を研究するためのモデルとして、成体ラットの下頸髄における同時後根挫滅損傷との直接の後根神経節（DRG）の注入を使用するプロトコルを提示します。 DRG感覚ニューロンは、周囲からCNSには、そのような触覚感覚や痛みなどの感覚情報を中継する責任があります。脊髄における感覚ニューロンの長い軸索投射は、長距離の軸索再生を研究するための良いモデルとなります。また、げっ歯類は、最小限の福祉合併症と後根挫滅損傷としての感覚経路の病変を生き残ることができるよう、研究者は完全に脊髄を病変部を必要とせずにCNSの軸索再生を学ぶことができます。四C5 - C8（頸部LEVEL 5から8）後根圧潰損傷は、前足の求心路遮断2のために有用なモデルであることが示されています。また、後根挫滅損傷は、それが、このようなグリア瘢痕形成など他の要因によって複雑ではないので、直接脊髄損傷よりも、軸索再生を研究する「クリーナー」モデルを提供します。 回生状態に神経細胞を再プログラムするウイルス遺伝子治療の使用は、ますます多くの神経学的状態3のための有望な治療戦略と考えられてきました。研究は、行動回復4、5、6とロバスト軸索再生を達成することができる成長促進タンパク質の導入遺伝子を担持するアデノ随伴ウイルス（AAV）ベクターの適用を示しています。免疫応答およびニューロンなどの非分裂細胞を形質導入する能力を誘発におけるAAVの見かけの低病原性は、作成しますその遺伝子治療のための最適のベクター。また、組換えAAV形態は、治療のために使用されます。この形態では、それは、宿主ゲノム7にそのウイルスゲノムを統合するようなレンチウイルスのような他のウイルスベクターに比べて挿入突然変異誘発のリスクを低減することができません。これは、AAV遺伝子治療用途のための安全な選択肢となります。 DRGは、感覚ニューロンの細胞体が含まれているとして、勉強および/または感覚軸索再生を促進するための遺伝子治療用ウイルスの投与のための最も適切な解剖学的な目標です。異なるAAV血清型およびレンチウイルスを比較した研究では、AAV血清型5（AAV5）がDRG 8に直接注射したとき、少なくとも12週間の時間経過にわたってDRGニューロンを形質導入において最も効率的であることが示されました。さらに、AAVは、大径ニューロフィラメント200 kDaのような全てのDRGニューロンのサブタイプを、形質導入、40％以上の形質導入効率を達成することができます（NF200）陽性ニューロンと小径カルシトニン遺伝子関連ペプチド（CGRP） -またはイソレクチンB4（IB4）陽性ニューロン4,8。 DRG注射および後根挫滅損傷の手術手順としては非常に侵襲的で繊細で、我々はこの記事では、新しいユーザーは非常に効率的な方法で手順を学ぶために役立つと信じています。 C8後根挫滅損傷 - 同時C5とC7のDRGに - 本稿では、成人の4週間C6に対照ウイルスAAV5-GFP（緑色蛍光タンパク質）の注射後のラットからの代表的な結果を示しました。このモデルは、感覚軸索再生を促進するためのウイルス遺伝子療法の使用を調査している研究者のために特に適しています。

<table><tbody><tr><td>Fast Green FCF dye</td><td>Sigma-Aldrich</td><td>F7258</td><td>For visualizing colorless solution. Recommended concentration: 1%</td></tr><tr><td>Cholera Toxin B subunit</td><td>List Biological Laboratories</td><td>104</td><td>For anterograde axonal tracing. Recommended concentration: 1%</td></tr><tr><td>IsoFlo</td><td>Zoetis</td><td>115095</td><td>Inhalation anesthetic (active ingredient: isoflurane)</td></tr><tr><td>Baytril 2.5% injectable</td><td>Bayer</td><td>05032756093017</td><td>Antibiotic (active ingredient: enrofloxacin). Manufacturer&#039;s recommended dosage: 10 mg/kg</td></tr><tr><td>Carprieve 5.0% w/v</td><td>Norbrook</td><td>02000/4229</td><td>Analgesic (active ingredient: carprofen). Manufacturer&#039;s recommended dosage: 4 mg/kg</td></tr><tr><td>Lacri-Lube</td><td>Allergan</td><td>PL 00426/0041</td><td>Eye ointment</td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>Fine Science Tools</td><td>FST 12502-12</td><td></td></tr><tr><td>Friedman Pearson Rongeur Curved 0.7 mm Cup</td><td>Fine Science Tools</td><td>FST 16121-14</td><td></td></tr><tr><td>Bonn Micro Forceps</td><td>Fine Science Tools</td><td>FST 11083-07</td><td>For performing dorsal root crush injury</td></tr><tr><td>Tissue Separating Scissors</td><td>Fine Science Tools</td><td>FST 14072-10</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>FST 14058-11</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine Science Tools</td><td>FST 11018-12</td><td></td></tr><tr><td>Goldstein Retractor</td><td>Fine Science Tools</td><td>FST 17003-03</td><td></td></tr><tr><td>Vannas Spring Scissors (straight)</td><td>Fine Science Tools</td><td>FST 15018-10</td><td></td></tr><tr><td>SURGIFOAM Absorbable Gelatin Sponge</td><td>Ethicon</td><td>1972</td><td>For bleeding control</td></tr><tr><td>Microliter Syringe RN701 (10 &amp;mu;L)</td><td>Hamilton</td><td>80330</td><td></td></tr><tr><td>Custom-made Removable Needle (for DRG injection)</td><td>Hamilton</td><td>7803-05</td><td>33 gauge, 38 mm, point style 3</td></tr><tr><td>Custom-made Removable Needle (for CTB injection)</td><td>Hamilton</td><td>7803-05</td><td>33 gauge, 10 mm, point style 3</td></tr><tr><td>UltraMicroPump with SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-1</td><td></td></tr></tbody></table>

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.

表現として、添付のDRGと横脊髄部C7に直接、DRGニューロンを形質導入し、四週間対照ウイルス、AAV5-GFPを注入した後に、脊髄における感覚軸索をトレースにこのプロトコルの有効性を示すために提示されています後根圧潰損傷（ 図1A）ことなくDRG。脊柱及び脊髄後角の両方における軸索はGFP（ 図1B）、ならびに注入DRG（ 図1C）内の...

Watch this Scientific Journal Video about Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration at JoVE.com

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

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 ...

dorsal-root-ganglion-injection-dorsal-root-crush-injury-as-model-for

Research

JoVE Journal

Neuroscience

17.8K ビュー.  University of Cambridge. 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.

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

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 ...

神経科学

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. ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工学

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

負傷ラット脊髄を修復するために末梢神経移植とマトリックスモジュレーションを組み合わせ

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

生物学

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

根神経節ニューロンと差別化された脂肪由来幹細胞を背：<em&gt;インビトロ</em末梢神経再生を研究するために&gt;共培養モデル

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

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

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

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

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

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 ...

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

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

<meta charset="utf8"></head><body>網膜神経節細胞軸索を標的とする軸索再生と介入を評価するためのラットの鞘温存視神経切断

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

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

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