Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

この作業は、国立難聴者協会のR01DC015824（KMS）およびT32DC00038（SDサポート）、防衛省のW81XWH-15-1-0472（KMS）、Bertarelli財団（KMS）、ナンシーセイレスデー財団（KMS）、ラウアー耳鳴り研究センター（KMS）などがあります。 Jessica E. Sagers氏に感謝の意を表します。

この研究プロトコールは、マサチューセッツ州の眼および耳の施設動物管理および使用委員会（IACUC）によって承認された。実験は世界医師会の倫理規定に従って実施された。 1.解剖の準備<ol><li> 手術テーブルの準備 <ol><li> 70％エタノールを使用して外科用テーブルを消毒する。 </li><li> 2つの非滅菌準備パッドを顕微鏡の隣に置きます。 </li><li>熱殺菌した手術用ハサミ、メスハンドル、マイクロナイフ、鉗子（＃4と＃55の2つずつ）を含む計器トレイを準備します。器具トレイと50mmの透明壁のガラス底ペトリ皿を1つの非滅菌パッドに置きます。 </li><li>無菌の＃15メス刃を得る。密封可能なプラスチック製の袋または容器（制度上のガイドラインに沿って死体を処分するため）。バケツの氷;冷たい滅菌ハンクス平衡塩類溶液（HBSS）を含む。これらのアイテムを他の非滅菌パッド。 </li><li> HBSSを50 mLのプラスチック試験管に移し、氷上に置きます。 </li></ol></li><li> 文化の準備 <ol><li>すべての材料を層流フードで準備する。 4つの標本ごとに、35mm4ウェルペトリ皿の4つのインレーに4つのオートクレーブ処理した10mmの丸いガラスカバースリップを入れる。 </li><li> 4つの標本ごとに、以下のものからなる300μLの全量を微量遠心チューブに混合する：組織接着剤10μL、NaHCO 3285μLおよびNaOH5μL。 </li><li>得られた溶液45μLをすべてのカバースリップに入れ、室温で少なくとも20分間放置する。この溶液は数時間カバースリップ上に放置することができるが、O / Nを放置することはできない。 </li><li>コンタミネーションに対する2つの障壁を作り出すために、1つまたは2つの35mm 4穴ペトリ皿を10cmのペトリ皿の中に置きます。 </li><li> 98％のダルベッコ改変イーグル培地（DMEM）、1％N-2、および1％アンピシリン（（マイクロ）遠心分離管内で1ウェルあたり65μL）で洗浄した。使用前に25℃に加温してください。 </li></ol></li></ol> 2.マウスの蝸牛外植片の解剖<ol><li> 新生児マウスの断頭 <ol><li> 60 mmのプラスチックペトリ皿を用意し、蓋に70％エタノールをスプレーして、表面が濡れるようにします。 </li><li>冷たいHBSSをプラスチック製の実験室チューブから60mmのプラスチックの底部と50mmの透明なガラス底のペトリ皿に注ぎます。解剖されていないときはいつでも組織を冷たく保つために、プラスチック実験室管と両方のペトリ皿を氷上に保管してください。 </li><li> P3-P5歳の新生児マウスをラテックス手袋の切れた指の氷上に置き、低体温が意識消失（約1〜3分）を引き起こすまで待つ。 </li><li>操作はさみでマウスをすばやく断頭し、シール可能なビニール袋に体を処分する。切断された新生児の頭を60mmのプラスチックシャーレの蓋の中の70％エタノール。 </li></ol></li><li> 側頭骨の巨視的切開 <ol><li>メスの刃を使用して、前端から後端（矢状縫合の上に直接）から表面切開を作ることによって、頭蓋骨の皮膚を除去する。 </li><li>外耳道の両方をメスの刃で切って、皮膚を前方に折り畳んで頭蓋全体を露出させます。 注：このステップには通常、解剖顕微鏡は必要ありませんが、視覚化を改善するために使用することができます。 </li><li> ＃15メス刃を使用して矢状縫合に沿って頭蓋を開きます。蝸牛の圧迫を避けるために、刃を前方から後方へ静かに上下に動かして頭蓋を切断してください。 注：頭蓋骨はこの年齢で骨化してはならず、簡単に切るべきです。 </li><li> ＃15メスを使用して、軌道の直後の垂直カットを作成して、スナウトを取り出して破棄します刃。 注：頭蓋骨の後部にはまだ脳と蝸牛が含まれているはずです。 </li><li> ＃4鉗子で鈍的切開を行い、前脳、小脳、および脳幹を処分する。 </li></ol></li><li> 蝸牛の微視的抽出 <ol><li>スコープの下に鉗子を置き、照明と焦点を最適化することによって顕微鏡（倍率6.5倍）と光源を調整します。 </li><li>冷たいHBSSで満たされた60mmプラスチックペトリ皿に頭蓋の2つの半分を置きます。 </li><li>周囲の静脈動脈（側頭骨内の曲がりくねった動脈）に隣接していると同定できる蝸牛/ eを完全に露出させ、＃4の鉗子を用いて臓器を側頭骨から鈍く分離する。 </li><li>蝸牛を50mmの透明なガラス底のペトリ皿に移し、顕微鏡の下に置く。 </li><li>氷上に頭蓋の他の半分と60ミリメートルのプラスチックシャーレを置きます。 </&gt; <li>蝸牛を前庭系から取り出し、＃4の鉗子で蝸牛耳の嚢（典型的には軟骨性）を慎重に切開する。それ以降のすべてのステップで＃55鉗子を使用してください。 </li></ol></li><li> 組織処理の最終ステップ <ol><li>マイクロナイフまたは2つの鉗子を用いて、コルチ器官に付着した螺旋靭帯を残りの蝸牛および神経節から注意深く分離することにより、内耳組織の処理を続ける。 <ol><li>螺旋靭帯と有毛細胞領域との間の隙間のより暗くて半透明な領域に切断し、螺旋靭帯を最も基本的な側面で把握し、尖った方向に動いている間にゆるやかに引き離すことに続く。 </li></ol></li><li>検体を配置して、蝸牛の長手方向矢状図を取得し、マイクロナイフを用いて蝸牛を2〜3つの切片に切断し、これらの切片を頂端、（中央、）およびbアナルターン。ペトリ皿に水平に横たわることができる蝸牛外植片を達成するために、有毛細胞および神経突起の実際の層と上および下組織を除去する。 注記：培養皿への安定した接着のための適切なサイズは、1つの蝸牛ターンの約半分である。 </li><li>腹膜を静かに剥がして、コルチ器官のすぐ上の非常に薄い半透明の層である房膜を取り除く。 </li><li> Reissnerの膜をすぐにSGNよりも優れたものにし、両側に鉗子で触れ、剥がしてください。 注：横に位置する損傷した有毛細胞領域をマイクロナイフで切り取って取り除きます。 </li></ol></li></ol> 3.検体の培養プレートへの移送<ol><li> 4つのカバースリップから45μLの組織接着剤溶液を除去する。各カバースリップを50μLの滅菌H 2 Oで洗浄する。水を吸引する。 </li><li> 1 mLのピペットを拾います。試料がチップの内側に付着しないように、約120μLのDMEMでピペットチップを濡らします。 </li><li> 1 mLピペットを用いて検体を個別に移す。 HBSSで満たされた、小さくて透明なガラス底のペトリ皿から最大70μLをピペットで取り、4ウェルのペトリ皿に4つのインレイ（カバースリップで調製）の1つに液体と共に試料を置く。 </li><li> 標本の向きが正しいことを顕微鏡（倍率50倍）で確認します。胸膜が除去された有毛細胞の領域が上向きであることを確認する。モディベラのトリムされた基底部分と一緒に、基底膜が下向きになり、カバースリップに付着することを確認する。 <ol><li>標本が検査の際に上下逆さまになっている場合は、ピペットチップに残っているHBSSをインレイに入れ、正しい方向になるまでピースの位置を変えます。 NOTE：これは、退化につながる可能性のあるカバースリップからの構造的支持なしに有毛細胞が培地中に浮遊することを防止する。 </li></ol></li><li> 1 mLピペットを使用して過剰のHBSSを吸引する。 15秒待ちます。 </li><li>調製した暖かい培地（98％DMEM、1％N-2、および1％アンピシリン）60μLを検体に直接ピペットで加える。ピペットで検体に触れないように注意してください。内側および外側のペトリ皿カバーを交換し、37℃でO / Nをインキュベートする。 </li><li>すべてのストック溶液を適切な場所に戻し、死骸や残留廃棄物を処分し、施設ガイドライン（ 例えば、エタノールや次亜塩素酸塩を使用して）に従って手術テーブルを除染し、器具をきれいにしてオートクレーブし、 。 </li></ol> 4.最終的な培養培地および対象物質の追加注：蝸牛外植片は、10-16時間後に使用する準備ができている。 <ol><li>混合物を準備する97％DMEM、1％ウシ胎児血清（FBS）、1％N-2、および1％アンピシリン（マイクロ遠心管内のウェルあたり65μL;使用前に溶液を25℃に暖める）。 </li><li>該当する他の物質を、それぞれの治療グループのソリューションに追加します。蛍光物質（ 例えば、緑色蛍光タンパク質（GFP）発現ウイルス;最近の発表では、10 10 GCのアデノ関連ウイルス（AAV）を50μL中48時間使用した） 18 、光。 </li><li>孵卵器から蝸牛外植片を含むペトリ皿を移し、層流フードの下に置く。 </li><li>インレーからのピペットで古い培地（FBS無し）を吸引する。 </li><li>調製した温かい培養（処理）培地（97％DMEM、1％FBS、1％N-2,1％アンピシリン、および対象物質）1ウェルあたり60μLを添加する。 </li><li>ペトリ皿をインキュベーター（37℃）に戻します。 </li><li>ビューr細胞の移動、または蝸牛外植片の剥離の兆候を同定し、最大7日後に染色を実施するために、顕微鏡下で公式に観察する。 </li></ol> 5.免疫蛍光 - 1日目<ol><li>ブロッキング溶液（94％リン酸緩衝生理食塩水（PBS）、5％正常ヤギまたはウマ血清（2次抗体に応じてNGS / NHS）、および1％非イオン性界面活性剤（ 表の表を参照 ） （98.6％PBS、1％NHS、および0.4％非イオン性界面活性剤をそれぞれの抗体とともに含む）で洗浄した。 注：抗体には、1：400+（ミオシン7A、内外の有毛細胞用）およびマウス抗TuJ1 1：200+（β-チューブリン、SGN用）またはマウス（IgG1）抗マウス抗体（後シナプス密度）、およびニワトリ抗NF-H1：1000 +（ニューロフィラメント（neurofilament））、抗マウスモノクローナル抗体SGNおよび神経線維の場合）。 &quot;+&quot; means &quot;以上である必要があります。 </li><li>ピペットを用いて培地を吸引する。検体に触れないように注意してください。 </li><li>層流フードの下で、滅菌PBSで蝸牛外植片を2回すすぎ、各洗浄後に5分間シェイカー上に検体を置く。 </li><li>シェーカー（フードの下）で20分間4％パラホルムアルデヒド（PFA - CAUTION）に組織を固定する。 PFAを吸引する。 </li><li> 60μLのPBSを蝸牛外植片に塗布し、ペトリ皿をシェーカーに5分間入れる。 PBSを吸引する。 3回繰り返します。 </li><li>蝸牛外植片を、シェーカー上で30分間、調製したブロッキング溶液（94％PBS、5％NHS、および1％非イオン性界面活性剤）中に置く。 </li><li>蝸牛外植片をRTでのカウンター上の対応する一次抗体（使用前にボルテックス）O / Nを用いて調製ストック抗体溶液（98.6％PBS、1％NHS、および0.4％非イオン性界面活性剤）に入れる。 注：ペトリ皿をプラスチック製のラップ（またはアルミ箔）で囲まれた湿ったペーパータオルで包みますGFPを発現するウイルスなどの蛍光物質を含む）を含み、皿を湿潤に保ち、抗体溶液O / Nの蒸発を防止する。 </li></ol> 6.免疫蛍光 - 2日目<ol><li>ステップ5.1で4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）染色（300nM）を調製し、ストック抗体溶液を使用して一次抗体に相補的な正しい二次抗体混合物を得る。 （ 例えば 、ヤギ抗ウサギ488 1：200+およびヤギ抗マウス568 1：200+ ORヤギ抗マウス（IgG1）568 1：1000+、ヤギ抗マウス（IgG2a）488 1：500+、およびヤギ抗ニワトリ647 1：200+またはファロイジン568 1：200+（内外の有毛細胞用））。 </li><li>一次抗体溶液を吸引する。 </li><li> 60μLのPBSを蝸牛外植片に塗布し、ペトリ皿をシェーカーに5分間入れる。 PBSを吸引する。 3回繰り返します。 </li><li>蝸牛外植片を準備ストック抗体溶液（98.6％PBS、1％NHS、および0.4％非イオン性界面活性剤）にres（使用前にボルテックス）をカウンターで1.5時間インキュベートし、光から保護する。 </li><li>二次抗体溶液を吸引し、DAPI染色液（シェーカー上に1分間置いてから吸引する）で蝸牛外植片をすすいでください。 </li><li> 60μLのPBSを蝸牛外植片に塗布し、ペトリ皿をシェーカーに5分間入れる。 PBSを吸引する。 3回繰り返します。 </li><li>鉗子を使用して、4ウェルプレートから検体を含むカバーガラスを取り出し、蒸留水で満たされたレシピエントにそれらを浸し、ペーパータオルに垂直に接触させてカバースリップを乾燥させる。 </li><li>顕微鏡スライド上に曇り止め装着媒体の一滴を置き、カバースリップを反転させて、下向きの組織を培地に浸す。 </li><li>周りを覆って透明なマニキュアを使用してカバースリップをシールします（ガラスの下に試料を押しつぶすことなく）。乾燥した状態となるまで15〜20分間明かりから覆った部分にスライドを平らにしておきますそれらを箱に入れたり、共焦点顕微鏡の下で検査したりしてください。 注：蛍光染色は、スライドを4℃または-20℃で保存することで最もよく保存されます。 </li></ol> 7.共焦点イメージング<ol><li>神経突起およびSGNを含むCorti地域の器官の概要およびズームイン画像を取得します。 注：イメージングプロセスの標準設定：1,024×1,024ピクセル、400Hzの速度、フレーム平均3、20％の総合レーザー強度、通常20倍および63倍の油浸漬（潜在的に2倍デジタルズームと組み合わせる）。 405 5％DAPI、 &quot;スマートゲイン&quot; 766.8V、 &quot;スマートオフセット&quot; 0.0％〜488 15％のフルオレセインイソチオシアネート（FITC）、 &quot;スマートゲイン&quot; 622.2VのDAPI、Myo7A、およびTuJ1染色プロトコルのチャンネル設定が概説されています。 、 &quot;スマートオフセット&quot; 0.0％ - 561 13％テトラメチルローダミンイソチオシアネート（TRITC）、 &quot;スマートゲイン&quot; 562.4V、 &quot;スマートオフセット&quot; 0.0％。 </li><li>画像をZスタックにマージするには、ソフトウェアを使用してz軸投影を得る。 </li></ol>

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J., Olze, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salicylate+modulates+Hsp70+expression+in+the+explanted+organ+of+Corti.">Salicylate modulates Hsp70 expression in the explanted organ of Corti.</a> Neurosci Lett. 501 (2), 67-71 (2011).</li><li>Kao, S. 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=Loss+of+osteoprotegerin+expression+in+the+inner+ear+causes+degeneration+of+the+cochlear+nerve+and+sensorineural+hearing+loss.">Loss of osteoprotegerin expression in the inner ear causes degeneration of the cochlear nerve and sensorineural hearing loss.</a> Neurobiol Dis. 56, 25-33 (2013).</li><li>Jan, T. A., Chai, R., Sayyid, Z. N., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+LacZ-expressing+cells+from+mouse+inner+ear+tissues+using+flow+cytometry.">Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.</a> J Vis Exp. (58), e3432 (2011).</li><li>Haque, K. D., Pandey, A. K., Kelley, M. W., Puligilla, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+embryonic+mouse+cochlear+explants+and+gene+transfer+by+electroporation.">Culture of embryonic mouse cochlear explants and gene transfer by electroporation.</a> J Vis Exp. (95), e52260 (2015).</li><li>Parker, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> J Vis Exp. (36), (2010).</li><li>Mulvaney, J. F., Dabdoub, A. <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+time+lapse+imaging+of+mouse+cochlear+explants.">Long-term time lapse imaging of mouse cochlear explants.</a> J Vis Exp. (93), e52101 (2014).</li><li>Wang, Q., Green, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+neurotrophin-3+in+synapse+regeneration+by+spiral+ganglion+neurons+on+inner+hair+cells+after+excitotoxic+trauma+in+vitro.">Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro.</a> J Neurosci. 31 (21), 7938-7949 (2011).</li><li>Landegger, L. D., 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=A+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Dilwali, S., Landegger, L. D., Soares, V. Y., Deschler, D. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Factors+from+Human+Vestibular+Schwannomas+Can+Cause+Cochlear+Damage.">Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.</a> Sci Rep. 5, 18599 (2015).</li><li>Soares, V. 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=Extracellular+vesicles+derived+from+human+vestibular+schwannomas+associated+with+poor+hearing+damage+cochlear+cells.">Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.</a> Neuro Oncol. , (2016).</li><li>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> J Assoc Res Otolaryngol. 14 (3), 321-329 (2013).</li><li>Yuan, 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=Ouabain-induced+cochlear+nerve+degeneration:+synaptic+loss+and+plasticity+in+a+mouse+model+of+auditory+neuropathy.">Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.</a> J Assoc Res Otolaryngol. 15 (1), 31-43 (2014).</li><li>Fernandez, K. A., Jeffers, P. W., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+&amp;#34;recovered&amp;#34;+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in &amp;#34;recovered&amp;#34; ears.</a> J Neurosci. 35 (19), 7509-7520 (2015).</li><li>Barclay, M., Constable, R., James, N. R., Thorne, P. R., Montgomery, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+sensory+stimulation+alters+the+molecular+make-up+of+glutamatergic+hair+cell+synapses+in+the+developing+cochlea.">Reduced sensory stimulation alters the molecular make-up of glutamatergic hair cell synapses in the developing cochlea.</a> Neuroscience. , 50-62 (2016).</li><li>Zinn, E., 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=In+Silico+Reconstruction+of+the+Viral+Evolutionary+Lineage+Yields+a+Potent+Gene+Therapy+Vector.">In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.</a> Cell Rep. 12 (6), 1056-1068 (2015).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol Ther. 18 (1), 80-86 (2010).</li><li>Shu, 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=Identification+of+Adeno-Associated+Viral+Vectors+That+Target+Neonatal+and+Adult+Mammalian+Inner+Ear+Cell+Subtypes.">Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.</a> Hum Gene Ther. , (2016).</li><li>Kao, S. Y., Soares, V. Y., Kristiansen, A. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRAIL-DR5+pathway+promotes+sensorineural+degeneration+in+the+inner+ear.">Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear.</a> Aging Cell. 15 (2), 301-308 (2016).</li></ol>

著者は何も開示することはない。

研究者は、蝸牛外植物を含む実験を行う前に解剖技術を完璧にしなければならない。有毛細胞は、学習曲線の早期に行われる解剖の間に一般的に損傷を受け、その完全性のための特に問題のある瞬間は、安定した手、適切な道具および経験を必要とする体膜の除去である。時間と資源を節約するために、解剖顕微鏡の下で視覚制御を行うべきであり、潜在的に損傷した領域に注目すべきであ?...

感音神経障害（SNHL）は、内耳または上行する聴覚経路への損傷を反映する。難聴はヒトの最も一般的な感覚障害であるが、治癒療法はまだ存在していない2 。蝸牛や聴覚の脳幹インプラントは、重度から重度のSNHLの患者にある程度の聴覚を回復することができますが、これらの装置で提供される聴力は、特に聴覚の発声を理解したり、音楽を聴いたりする試みにおいて、 毛細血管変性は長い間、外傷性聴覚事象（ 例えば、大きな騒音への曝露）の主な結果と考えられているが、有毛細胞から聴神経へ情報を伝達するシナプスは少なくとも音響外傷3 と同様に脆弱であるという証拠が増えている3 、 4,5 </sup<html> &gt; 、 6 。聴覚機能の評価のための現行の金標準である人間の聴力検査閾値は、内耳の特定の細胞損傷を予測しないので、細胞変性をできるだけ早期に検出し、適切な治療を開始するためにより洗練されたツールが必要です。 難聴のための有望な薬学的治療は、しばしば、インビトロでの均一な細胞培養物について試験されるが、このようなシステムは、蝸牛の微小環境を正確にモデル化しない。蝸牛細胞は、Corti 10,11またはSpiral Ganglion Neurons（SGN） 12の臓器を単離培養したときに失われる重要なインビボ過程である蝸牛8,9内の他の細胞型に影響を及ぼす栄養因子を分泌することが知られている分子マーカーを分析するしかしながら、「精密医学」の追求において、難聴のための新たなパーソナライズされた治療法を確立するためのインビトロデータの検証に必要であり得るインビボ研究は、かなりの資源と時間を必要とする。聴力検査とその後の蝸牛全体装着の中耳または円形窓膜注入を完璧に行うためにどれだけの労力が必要ですか？蝸牛外植体として知られている器官型ex vivo培養物の有望な化合物の効率的なスクリーニングは、 14,15,16,17 。 この記事では、治療された蝸牛外植片を生成、維持、評価するためのプロトコルについて詳述します。スクリーニングでの使用を含むこのモデルの特定のアプリケーションが強調されています遺伝子治療のためのウイルスベクターの比較評価を含む。 エクスビボでの外植片アプローチにより、研究者は、異なるタイプの細胞集団に対する所定の治療の効果を現場で視覚化し、細胞型特異的機構の同定およびその後の標的化治療の洗練を容易にすることができる。 全体的に、この技術は、蝸牛内に共存する非常に異なる細胞タイプ間の重要なクロストークを維持しながら、 生体外で蝸牛を研究するモデルを提供する。

<table><tbody><tr><td>Ampicillin, Sodium Salt</td><td>Invitrogen</td><td>11593-027</td><td></td></tr><tr><td>anti-CtBP2 antibody, mouse(IgG1)</td><td>BD Transduction Laboratories</td><td>612044</td><td></td></tr><tr><td>anti-Myo7A antibody, rabbit</td><td>Proteus Biosciences</td><td>25-6790</td><td></td></tr><tr><td>anti-NF-H antibody, chicken</td><td>EMD Millipore</td><td>AB5539</td><td></td></tr><tr><td>anti-PSD95 antibody, mouse(IgG2a)</td><td>Antibodies Inc.</td><td>75-028</td><td></td></tr><tr><td>anti-TuJ1 antibody, mouse</td><td>BioLegend</td><td>801202</td><td></td></tr><tr><td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td><td>Corning</td><td>354241</td><td></td></tr><tr><td>CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>188161</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 100 x 20 mm</td><td>Greiner Bio-One</td><td>664160</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 35 x 10 mm, 4 inner rings</td><td>Greiner Bio-One</td><td>627170</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 60 x 15 mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>Clear Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&amp;reg;, Sleeve/20, 50 x 7 mm</td><td>Ted Pella Inc.</td><td>14027-20</td><td></td></tr><tr><td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13 - 0.16mm</td><td>Ted Pella Inc.</td><td>260368</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Distilled water, 500 mL</td><td>Thermo Fisher Scientific</td><td>15230-162&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose, pyruvate, no glutamine, 500 mL</td><td>Thermo Fisher Scientific</td><td>10313-039</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #55 Forceps (Dumostar)</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Ethyl alcohol (EtOH), Pure, 200 proof, anhydrous, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>459836-1L</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, USDA-approved regions, 500 mL</td><td>Thermo Fisher Scientific</td><td>10437-028&amp;nbsp;</td><td>Aliquot in 50 mL tubes and store in -20&amp;deg;C freezer</td></tr><tr><td>goat anti-chicken-647 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21469</td><td></td></tr><tr><td>goat anti-mouse(IgG)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-11004</td><td></td></tr><tr><td>goat anti-mouse(IgG1)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21124</td><td></td></tr><tr><td>goat anti-mouse(IgG2a)-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td></td></tr><tr><td>goat anti-rabbit-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>R37116</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, sterile, EmbryoMax Ultra Pure Water, 500 mL</td><td>EMD Millipore</td><td>TMS-006-B</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), calcium, magnesium, no phenol red, 500 mL</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Instrument Tray with Lid Stainless Steel</td><td>Mountainside Medical</td><td>TechMed4255</td><td></td></tr><tr><td>Micro (dissecting) knife &amp;ndash; angled 30&amp;deg;</td><td>Fine Science Tools</td><td>10056-12</td><td></td></tr><tr><td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&quot;), 1 mm thick, white, pack of 72</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>N-2 Supplement (100X), 5 mL</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, Sodium Bicarbonate 7.5% solution, 100 mL</td><td>Thermo Fisher Scientific</td><td>25080-094</td><td></td></tr><tr><td>NaOH, sodium hydroxide solution, 1 L</td><td>Thermo Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>Normal Horse Serum (NHS)</td><td>Invitrogen</td><td>16050130</td><td></td></tr><tr><td>Operating scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-6806</td><td></td></tr><tr><td>Paraformaldehyde (PFA), Reagent Grade, Crystalline</td><td>Sigma-Aldrich</td><td>P6148</td><td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td></tr><tr><td>PBS, pH 7.4, 500 mL</td><td>Thermo Fisher Scientific</td><td>10010-023&amp;nbsp;</td><td>Autoclave prior to use</td></tr><tr><td>Phalloidin, Alexa Fluor 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A12380</td><td></td></tr><tr><td>Prep Pad, Non Sterile&amp;nbsp;</td><td>Medline</td><td>05136CS</td><td></td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL</td><td>Eppendorf</td><td>022363719</td><td>Autoclave prior to use</td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td>Autoclave prior to use</td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>Stemi 2000-C Stereo Microscope</td><td>Zeiss&amp;nbsp;</td><td>000000-1106-133</td><td></td></tr><tr><td>TCS SP5 confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Triton-X (non-ionic surfactant)</td><td>Integra</td><td>T756.30.30</td><td></td></tr><tr><td>VectaShield antifade mounting medium for fluorescence</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Zipper Bag, Reclosable, 4 x 6&#039;&#039; - 2 mm thick</td><td>Zipline</td><td>0609132541599</td><td></td></tr></tbody></table>

neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

過去数十年間にわたり聴覚研究において顕著な進歩があったが、内耳の繊細な機械感覚構造の損傷または喪失を典型的に伴う状態である感音神経難聴（SNHL）の治療法はまだない。近年、精巧なin vitroおよびex vivoアッセイが出現しており、SNHLの治療法を開発するためのリソースを最小限に抑えながら、潜在的治療化合物のスクリーニングを可能にしています。特定の細胞型の均質な培養物が現在の研究において重要な役割を果たし続けているにもかかわらず、多くの科学者は現在、蝸牛外植片としても知られているより複雑な器官型の内耳細胞培養に頼っています。内耳内の組織化された細胞構造の保存は、内外の有毛細胞、らせん状神経節ニューロン、神経細胞を含む蝸牛インフルエンザの様々な成分のin situ評価を容易にするites、および支持細胞。ここでは、新生仔ネズミの蝸牛外植片の調製、培養、処置、および免疫染色を提示する。これらの外植片の注意深い準備は、SNHLに寄与する機構の同定を容易にし、聴覚研究共同体にとって貴重なツールとなる。

多くのプロトコルがCorti外植片の器官に焦点を当てていますが、この技法はSGNを含む蝸牛ターン全体の解剖学的構造を保存しようとします。これにより、研究者は、Cortiの器官に加えて、SGNの神経突起および体細胞に対する所与の治療の効果を分析する機会を得ることができる。ここに記載されているように、モディオラスの一部を保存する解剖を行うことは、コルチ...

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聴覚研究における体外スクリーニングツールとしての新生児マウス蝸牛外植片技術 |医学 |ビデオ

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

このプロトコールの目的は、新生仔ネズミの蝸牛外植片の調製、培養、処置および免疫染色を実証することである。この技術は、聴覚研究におけるインビトロスクリーニングツールとして利用することができる。

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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Sensory Systems

SCIENTISTS IN ACTION

17.5K ビュー.  Massachusetts Eye and Ear. このプロトコールの目的は、新生仔ネズミの蝸牛外植片の調製、培養、処置および免疫染色を実証することである。この技術は、聴覚研究におけるインビトロスクリーニングツールとして利用することができる。 

ビデオ: Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAPのテストバッテリー：リスニングの意味を理解する

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

マウス蝸牛外植片の長期的タイムラプスイメージング

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

神経科学

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 ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

大人のマウスにおける人工内耳表面製剤

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

コルチの器官に向かう細胞の移動を探るインビトロタイムラプスライブセルイメージング

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

In vitroモデルとしての新生児蝸牛外植片全体

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

マウス蝸牛有毛細胞の効率的なIn vitro培養

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

著者スポットライト:聴覚研究のためのマウス有毛細胞の培養における進歩 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

新生仔マウスの初代蝸牛有毛細胞の単離と培養

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...

新生児マウス蝸牛移植法

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