Name: surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane
Uploaded: 2015-03-16T14:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

注：すべての手順や動物の取り扱いは、NIHの倫理ガイドラインを遵守し、カリフォルニア大学の制度的動物実験委員会、サンフランシスコのプロトコル要件を承認した。 1.手術のための動物の準備<ol><li>きれいな、専用スペースでの外科的処置を行ってください。手術前にガラスビーズ滅菌器で滅菌する、すべての手術器具をオートクレーブ。 注：このプロトコルでは、生後10〜12（P10-12）FVBマウスを使用しています。異なる年齢のマウスの株は、特定のプロジェクトのニーズを満たすために使用することができる。水疱の骨が難しくなりますので、P40より古いマウスは、挑戦することができます。 </li><li>塩酸ケタミン（100mg / kg）の混合物の腹腔内注射、塩酸キシラジン（10mg / kgの）、およびアセプロマジン（2mg / kgの）でマウスを麻酔。動物は、もはやそのようなつま先のピンチのような痛みを伴う刺激に応答した後にのみ、外科準備を開始します。もしnecessaryは、オリジナルの麻酔薬平面を復元するために麻酔薬カクテルのブースター用量（五分の一のオリジナル用量）を管理する。 注：この年齢で観測された死亡率のほとんどは麻酔薬の過剰投与によって引き起こされるので、この段階では注意してください。 </li><li>動物のまばたき反射を抑制し、麻酔中湿った目を保つために、保護眼軟膏で動物の目をカバーしています。 </li><li>マウスが完全に目を覚ましになるまで、麻酔後の低体温を防ぐため、作業中は加熱パッド上に拡張され、首にマウスを置きます。 </li><li>バリカンで左ポスト耳介地域を剃ると外科的操作の前に70％エタノール及びポビドンヨードで消毒する。 </li></ol> 2.手術手順とベクター注入<ol><li>鼓室胞を露出させるためにポスト耳アプローチを使用してください。ポスト耳介筋肉を露出させるために、小さなハサミで皮下組織を切開。目に脂肪組織を後退後切開部の電子後方側は、側頭骨を露出させるために切開に垂直な左右の筋肉を分離する。この切開は十分な可視化と快適に作業するのに十分な長さであることを確認してください。 </li><li> 25 G針で鼓膜胞を穿孔し、蝸牛の基底回転へのアクセスを許可するように戻って骨を剥離することにより、鉗子で、必要に応じて穴を展開し、[あぶみ骨動脈と丸い窓膜を可視化するために十分に広げる（RWM） 。 </li><li>ホウケイ酸キャピラリーピペットで中央に優しくRWMをパンク。この時点ではRWMを通して流体流出を観察する。これは正常です。流出が安定するまで待ちます（蝸牛から流出した流体は、滅菌濾紙で乾燥させる）。 注：保持とRWMの穿孔ができるだけ小さくなるように、ガラス針を進める際には注意してください。使用される顕微鏡に応じて、ピペットがピペットを用いてマイクロマニピュレーターを用いて、または手で保持するホルダー。 </li><li>パッチクランプピペットが用意されているのと同じ方法プラーピペットを用いて注入するためのピペットを準備します。内と外のウイルスをピペットが簡単に行われますように（直径約15μm​​）先端径が大きいことを確認してください。 </li><li>インジェクションピペットにAAV1-VGLUT3の1から2μL、AAV1-GFPまたはAAV2-GFPを策定する。流出（5〜10分）、安定化、microinject同じ穴を通して鼓室階にこの固定ボリュームは、以前RWMに行われた後。 </li><li>筋肉の小さなプラグをピペットを引き出した後、すぐにRWのニッチを密封し、ラウンド窓からの漏れを回避するために、筋肉に置かれた組織接着剤の小滴で固定します。 注：非常によく、針が蝸牛-このステップからの外リンパの漏出を防止するために、筋肉の小さなプラグを除去した後、この穴をシール聴力を維持することが重要です。完全にRWMをシールに失敗すると、時間をかけて難聴になります。 </li><li>注射後、カバー脂肪組織と聴覚の水疱の穴、彼らの通常の位置にポスト耳介筋や脂肪組織を返し、6-0または小さい吸収性クロム縫合糸で層に傷を縫合。 </li><li>ポビドンヨードと傷口を消毒する。 </li></ol> 3.術後ケア<ol><li>暖かいケージにマウスを置き、彼らが完全に戻っ母親とそれらを移動回収されるまで、それらを無人のままにしないでください。それは、母親と一緒に戻って子犬を置く前に、ケージから男性を削除することを推奨します。 </li><li>炎症および疼痛を管理するために、3日間その後術後鎮痛と、24時間の皮下カルプロフェン（2ミリグラム/ kg）を投与する。苦痛、異常な体重減少、痛み、または感染の徴候について毎日動物を監視します。一般的に、すべてのマウスが正常に機能するべきである手術後3 日目による。苦痛や病気の徴候は3日後にマウスに表示された場合は、Tを安楽死を検討施設のガイドラインに従って、彼はマウス。 </li></ol>聴覚脳幹反応（ABR）録画を用いてウイルス送達後の蝸牛機能4.評価 NOTE：聴性脳幹反応（ABR）は、聴覚が潜在脳における進行中の電気的活動から抽出され、頭皮の下に配置された電極を介して記録された誘発である。動物は、音で刺激される。結果の記録は、聴覚刺激の発症後最初の10ミリ秒で聴覚路に連続する点の電気的活動を反映して5波で構成されています。 <ol><li>このプロトコルのセクション1.2で説明したようにアセプロマジンなし除き、塩酸ケタミンとキシラジン塩酸塩の混合物の腹腔内注射によりマウスを麻酔。 </li><li>マウスが完全に目を覚ましになるまで、麻酔後の低体温を防ぐために、聴力検査を通じて加熱パッド上にマウスを置きます。 注：使用この実験では、次の音の刺激：（5ミリ秒の期間; 31 Hzのプレゼンテーション率）をクリックします。 </li><li>頂点で、左耳（参照）の耳介の下、および対耳（グランド）の下に皮下針電極を配置し、以前に防音室27,28で説明したようにのABRの記録を開始。ダウン最大振幅から5デシベルの音圧レベル毎のレコードABR波形。 </li><li>早けれウイルス送達後4日間などの術後のABR閾値を決定 注：閾値は波の応答のピークI-Vは、明らかに繰り返し目視上に存在していた最低の刺激レベルとして定義される。 </li><li>私は蝸牛神経からのアクティビティを分析するために波を測定します。検出可能なABR波形が得られる最低刺激レベルを閾値として定義される。 </li></ol>免疫蛍光を用いて5蝸牛導入遺伝子タンパク質発現<ol><li> intrapの過剰摂取によりマウスを麻酔塩酸ケタミンとキシラジン塩酸塩の混合物のeritoneal注入アセプロマジンなしを除いて、このプロトコルのセクション1.2で説明したように。 </li><li>動物は、もはやそのようなつま先のピンチのような痛みを伴う刺激に応答した後にのみ頭を首を切る。 </li><li> 26を説明するように全体のマウント免疫蛍光のためにアウトとプロセス蝸牛を解剖。抗VGLUT3および抗ミオシン7aの抗体を用いて蝸牛全体マウント免疫蛍光法の詳細なプロトコルはアキールら 、2013 29に記載されている。 </li><li> 250希釈：GFP標識のために、1でウサギ抗GFP抗体を用いて4℃で一晩蝸牛全体のマウントをインキュベートする。 </li><li> PBSで10分間、二回蝸牛をリンスした後、Cy2とコンジュゲートしたヤギ抗ウサギIgGで2時間インキュベート1に希釈した：PBSで4000。 </li><li> 10分間、2回PBSで蝸牛をすすぎ、15分間DAPIでそれらを培養する。 </li><li>スライドガラス上に蝸牛をマウントし、ウントを観察共焦点免疫蛍光顕微鏡えー。 </li></ol>

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A., 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=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+Ear+Transgene+Expressionafter+Adenoviral+Vector+Inoculation+in+the+Endolymphatic+Sac+Hum.">Inner Ear Transgene Expressionafter Adenoviral Vector Inoculation in the Endolymphatic Sac Hum.</a> Gene Ther. 10 (5), 769-774 (1999).</li><li>Praetorius, M., Baker, K., Weich, C. M., Plinkert, P. K., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+preservation+after+inner+ear+gene+therapy:+the+effect+of+vector+and+surgical+approach.">Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach.</a> ORL J. Otorhinolaryngol. Relat. Spec. 65 (4), 211-214 (2003).</li><li>Carvalho, G. J., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cochleaostomy+and+intracochlear+infusion+on+auditory+brain+stem+response+threshold+in+the+guinea+pig.">The effect of cochleaostomy and intracochlear infusion on auditory brain stem response threshold in the guinea pig.</a> Am. J. Otol. 20 (1), 87-90 (1999).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. K., 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=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

本研究では、遺伝的欠陥によって損なわれている通常の聴覚機能を回復または救助を目的とし、詳細に蝸牛遺伝子治療に使用することができる技術が記載されている。それは典型的に非外傷性であるように、このアプローチは、人工遺伝子導入または他の潜在的な分子の治療法30に安全である。蝸牛療法のための他のアプローチは、腹側のアプローチ24、マウスおよびモルモ?...

遺伝子治療は、長い遺伝性難聴のための潜在的治療法として提案されているが、この分野での成功は、1とらえどころのないままである。現在までに、ウイルス媒介性の方法論は、比較的アクセス蝸牛内の特定の細胞型を標的とする理論的能力により優勢いる。アデノウイルス（AV）、アデノ随伴ウイルス（AAV）の両方が蝸牛遺伝子送達のために使用されている。のAAVは、多くの理由のために蝸牛で有利である。彼らは、複製欠損ウイルスであると効率的にニューロン、難聴の原因の数の重要な標的を含む、異なる種類の細胞に遺伝子導入する分子を転送することができます。細胞へのAAVのエントリは、特定の受容体2によって媒介される。このように、特定の血清型の選択は、形質導入される細胞型に適合しなければならない。のAAVは、効率的に有毛細胞3をトランスフェクトし、トラヒックの安定した長期発現をもたらす、宿主ゲノムに組み込むことができnsgenicタンパク質や細胞4における表現型の変化。毛髪細胞再生短期用途には必ずしも有利ではないが、長期発現は、遺伝的欠陥を安定救助のために非常に重要である。 AAVは、任意のヒト疾患または感染に関連しない耳毒性5,6,7を示さないれていないので、彼らは損失8聴力の遺伝型の遺伝子治療に使用するための理想的な候補である。 ウイルスベクターを用いて哺乳動物の内耳への外因性遺伝物質の移動は、過去10年間研究されており、損失9聴力の遺伝的および後天両方の形態を治療するための有望な技術として浮上している。蝸牛は、潜在的にいくつかの理由のために、遺伝子治療のための理想的な標的である：1）その小さな容積は、必要なウイルスの限定された量を必要とする。他の器官系の制限副作用2）の相対的な単離; 3）その流体充填チャンバは、ウイルスを容易にする迷路10、11,12,13,14、15を通じて配信。 先天性難聴のマウスモデルは、系統的、複製可能な方法で、内耳の開発を監視するための研究の多くの方法の使用を可能にする。マウス蝸牛の小さいサイズは、いくつかの外科的困難を提示しませんが、マウスが他の種16に比べていくつかの実験的な利点で、遺伝性難聴の研究に極めて重要なモデルとなる。マウスモデルは、遺伝的連鎖解析、詳細な形態学的観察の収集、および病原性のシナリオをシミュレートを通して特性の範囲の評価を可能にする。のような、それらがウイルス媒介遺伝子治療のための良好な候補である。技術の進歩と組み合わせたマウスでの広範な遺伝学的研究は研究室17,18、19、20,21全体で再現可能な方法で、遺伝子改変されたマウスを作製することが可能になりました。 Furthermor両方を取得し、この動物モデル22、23,24で厳密なテストを可能にする、マウスにおける難聴表現型を継承するための電子、多数のモデルが存在する。従って、マウスモデルにおいてウイルス媒介遺伝子治療を用いて聴覚を補正するヒト疾患の治療法の探索において適切な最初のステップである。 我々は以前胞性グルタミン酸輸送体3（VGLUT3）を欠くトランスジェニックマウスによるIHCリボンシナプス25におけるグルタミン酸放出の欠如のために聴覚障害者が生まれていることが示されている。この変異は感覚毛細胞の一次変性につながらないので、これらの変異マウスは、潜在的に先天性難聴のための蝸牛遺伝子治療をテストするに優れたモデルである。 現在までに、蝸牛遺伝子治療のためのウイルス送達技術の数が正円窓膜拡散、正円窓膜注射、および蝸牛を介した送達を含む、記載されている。強力ありますIAL利点及びこれらのアプローチ9のそれぞれの短所。 ここでは、正円窓膜（RWM）を介してVGLUT3 KOマウス内耳へのウイルス媒介遺伝子送達のための外科的方法を報告している。耳介RWM注入法は、優れた聴覚保護と低侵襲性であり、かつ比較的高速である。我々は以前に公開しているように、このマウスモデルで聴力回復するための努力で、VGLUT3遺伝子（AAV1-VGLUT3）を運ぶAAV1ベクターは、生後12日目にこれらの聴覚障害のマウスの蝸牛に導入した（P！@）、その結果26聴力の回復。導入遺伝子のタンパク質発現を免疫蛍光（IF）を使用して確認しながらVGLUT3 KOマウスにおいて聴力は、聴性脳幹反応（ABR）によって確認した。この方法論は、このように、ウイルス媒介遺伝子治療は、そうでなければ難聴になるだろう遺伝的欠陥を修正することができることを実証している。

<table><tbody><tr><td>Ketamine</td><td>Butler Schein</td><td></td><td></td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td></td></tr><tr><td>Acepromazine</td><td>Provided by UCSF LARC</td><td></td><td></td></tr><tr><td>Carprofen analgesia</td><td>Provided by UCSF LARC</td><td></td><td></td></tr><tr><td>Betadine</td><td>Betadine Puredue Pharma</td><td></td><td></td></tr><tr><td>dexamethasone ophthalmic ointment (TobraDex)</td><td>Alcon</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Braintree scientific, inc.</td><td></td><td></td></tr><tr><td>25G needle</td><td>BD</td><td>305127</td><td></td></tr><tr><td>Borosilicate capillary pipette</td><td>World precision instruments, inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Suture PDS*plus Antibacterial</td><td>Ethicon</td><td>PDP149</td><td></td></tr><tr><td>Tissue glue (Vetcode)</td><td>Butler Schein</td><td>31477</td><td></td></tr><tr><td>Rabbit Anti-GFP antibody</td><td>Invitrogen</td><td>A11122</td><td></td></tr><tr><td>Dissecting microscope&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Leica</td><td></td><td>MZ95</td></tr><tr><td>Flaming/ Brown Micropipette&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sutter Instrument Co</td><td></td><td></td></tr><tr><td>Puller Model P-97&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>TDT BioSig III System&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;Tucker-Davis Technologies</td><td></td><td></td></tr></tbody></table>

surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

感音難聴からの機能的回復を達成するために使用される遺伝子治療は、難聴に寄与する根本的な分子的および遺伝的メカニズムのより良い理解を与えることを約束する。内耳へのベクターの導入は、既存の構造への損傷を最小限に抑えながら、広く蝸牛全体にエージェントを配布する方法で行われなければならない。この原稿は、分子、薬理学的、およびマウス生後10日目と丸い窓膜（RWM）を介して、古いのウイルス送達を用いてマウス蝸牛治療に用いることができる後の耳介の外科的アプローチについて説明します。血液の損失を最小化し、動物の死亡を回避しながら、この外科的アプローチは、鼓室階への迅速かつ直接の受け渡しが可能になります。完全に聴力を維持しながら、この手法は、無視できるか、インナーと中耳の本質的な構造への損傷だけでなく、首の筋肉を伴う。この手術法、水疱glutamの有効性を実証するために、食べトランスポーター3ノックアウト（VGLUT3 KO）マウスは、アデノ随伴ウイルス（AAV-1）を使用して内耳にVGLUT3の送達後に聴力回復先天性難聴のマウスモデルの例として使用される。

蝸牛分子治療のための技術的特徴とポスト耳アプローチの有用性を確認するには、AAV1-VGLUT3、AAV1-GFPおよびAAV2-GFPはRWM経由P10-12マウス内耳に送達された。このアプローチは、内有毛細胞内で正常に導入遺伝子発現（IHC）（VGLUT3 図1及びGFP 図2及びGFP 図3A）、外有毛細胞（OHC）（GFP 図2）および支持細胞（GFP、図2および図3A?...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

円窓膜を介してマウスインナーイヤーにウイルスにより媒介遺伝子送達のための手術方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.4K ビュー.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

ビデオ: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

発生生物学

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

成人および新生児マウス内耳への薬剤の局所投与への外科的アプローチとしての Canalostomy

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

遺伝学

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

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

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

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

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

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

神経科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

聴覚路の光遺伝学的刺激のためのマウス背側蝸牛神経核の直接可視化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

マウス網膜色素上皮における遺伝子治療用ウイルスベクターの縁アプローチ、網膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

蝸牛細胞の変性を誘導するための迅速かつ信頼性の高いテクニック：耳毒性剤送達のためのマウスラウンドウィンドウアプローチ

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

医学

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

ヒト以外の霊長類コモンマーモ (用いた) で内耳に薬の管理術

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

新生児マウス内耳遺伝子デリバリーの後半規管アプローチ

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

マウス内耳に標的薬の配布方法の比較研究：Bullostomy<em&gt;対</em&gt; Transtympanicインジェクション

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

円窓膜を介してマウスインナーイヤーにウイルスにより媒介遺伝子送達のための手術方法

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