この資料で説明する作業は、NCI RO1 CA166907、NIDCD RO1 DC 002396 と RO3 DC011621 によって支持されました。

雄 Wistar ラットは、国立衛生研究所動物に従って処理されたガイドラインおよび南イリノイ大学学校の医学研究所動物ケアおよび使用委員会承認プロトコルを使用します。トランス鼓膜薬物送達の効果を検証する中薬管理および後 72 時間前に麻酔下でラットの聴性脳幹反応 (ABR) を行った
1. 聴性脳幹反応 (ABR)
注:ABR の測定は、聴覚テスト装置とソフトウェアを使用して収集されました。ABR 誘発電位または第 8 脳神経高周波波を表します (I および II を波) および他の高い聴性脳幹構造提案蝸牛神経核 (波 III)、横帯 (波 IV) 劣っているなど丘 (V 波)。これらの波は、レーテンシーによって区別されます。ABR の測定は12を前述のように行った。
<ol><li>混合物 90 mg/kg 17 mg/kg とケタミン キシラジンを介して腹腔内投与のラットを麻酔します。つま先ピンチ反射を介して麻酔の深さを確認します。目の乾燥を防ぐ、麻酔中に潰瘍を避けるために両方の目に点眼潤滑 (または鉱物油滴) を適用されます。</li>
	<li>腹臥位制御音響ブース内部加熱パッド (37 ° C) でラットを配置します。聴覚検査機器は外、ブースの横にあります。</li>
	<li>それに応じてステンレスの電極を挿入: リアの接地電極の側面、頭蓋骨の上に直接 2 つの耳とそれぞれの耳の耳介の下否定的な電極の間肯定的な電極。</li>
	<li>8、16、32 kHz で 5 ms トーン バーストとして高周波数探触子を用いた音響刺激を適用します。デシベル音圧レベル (dB SPL) として刺激強度を決定します。これは 10 dB SPL で始まり、10 dB ステップ サイズの 90 dB SPL に達する。インパルス精密サウンド レベル メーターを使用して 90 dB SPL の最大出力に音の強度を調整します。 注:テスト結果は、末梢聴覚器官、蝸牛、および前述の聴覚の構造の整合性の指示を与えます。波形は入力された刺激の 15 ms 以内で生成され、波の遅延時間依存伝導時間を順番に 3 つの重要な要因によって規制されている: 脳と、強度と音の周波数のボリューム。聴覚誘発電位は、製造元によって提供されるソフトウェアを使用して記録しました。</li>
	<li>目立つようにしきい値として 0.5 μ V の振幅の 2 つの異なる波形 (II、III) を呼び起こすことができる最低の強度を検討します。 注:しきい値シフトは、治療前に得られた閾値と比較して治療後の測定閾値の差を表します。</li>
</ol>2. トランス鼓膜注射
<ol><li>トランス tympanically 薬を管理するには、左側臥位でラットを配置します。<ol>
<li>2.5 mm 使い捨て耳アジマスを外耳道に挿入します。</li>
		<li>手術範囲を使って、鼓膜が表示されるよう、鏡を位置します。</li>
		<li>29 X *、0.5 mL インスリン注射器を使用して [R] - N - フェニル イソプロピル アデノシンの 50 μ L を描画 (R-PIA) ソリューション (1 μ M)、8-シクロペンチルホモログ-1, 3-dipropylxanthine (みた) ソリューション (3 μ M)、または siRNA ソリューション (0.9 μ g) (5 単位) を注入します。</li>
		<li>鼓膜の下前歯に針を直接に、鏡を使用します。</li>
		<li>針で細胞膜に穴を開けないし、1.2.3 に記載されている薬の管理します。15 分のこの位置に残りの部分にラットを許可します。 注: 50 μ L は、鼓膜の後ろに合わせて十分な量をする必要があります。投与後、耳の三半規管の流体必要がないです。</li>
		<li>右側臥位置し、手順 1.2.5 を通じて 1.2.1 他耳の管理のためにラットを配置します。</li>
	</ol>
</li>
	<li>シスプラチンの腹腔内受信、ラットのラットを 37 ° C で加熱パッドの仰臥位にします。<ol>
<li>21 X 3/4 を使用して蝶の針 (12"長さチューブ)、シスプラチン (11 mg/kg ・ 1 mg/mL 滅菌リン酸バッファー生理食塩水 [PBS] ソリューション) を描画します。</li>
		<li>シリンジ ポンプを使用して、シスプラチン (1 mg/mL)を介して腹腔内投与 30 分以上を管理します。 注:250 g ラット ボリュームは 1 分あたり約 0.1 mL の割合で 2.75 mL になります。</li>
	</ol>
</li>
	<li>これらの手順の中で麻酔の深さを監視し続けます。シスプラチン投与が完了すると、その呼吸を妨害することはないことを確かめて、腹臥位でケージに戻ってラットを配置します。</li>
	<li>完全に回復するまでのラットを監視します。</li>
</ol>3. 蝸牛郭清と脱灰
<ol><li>次の (72 h) 後最終的な ABR、混合物 90 mg/kg 17 mg/kg とケタミン キシラジンを介して腹腔内投与のラットを麻酔します。つま先ピンチ反射と麻酔を確認します。斬首を介してラットを安楽死させます。</li>
	<li>13を前述のように側頭骨を分析します。</li>
	<li>(完全に覆う蝸牛) 7 mL ガラス シンチレーション バイアル PBS 溶液 × 1 で 4% パラホルムアルデヒドに蝸牛を配置します。4 ° C で一晩冷蔵庫で冷やしますパラホルムアルデヒドを削除し、室温で 1 x PBS で洗浄します。</li>
	<li>PBS を取り外して、EDTA (pH 7.3) の 120 ミリメートルのソリューションで完全に管を埋めます。室温で回転子に置き、EDTA 溶液を毎日変更する 2 〜 3 週間の脱灰処理を許可します。</li>
</ol>4. セクショニング
<ol><li>脱灰処理の完了時に完全に 4 ° c: 10% ショ糖溶液、20% ショ糖 20% ショ糖と最適の切削温度 (OCT) 化合物の埋め込みの 1:1 混合物で 24 時間以下のソリューション (7 mL) に蝸牛を浸します。</li>
	<li>新鮮な 10 月場所を埋めるし、15 mm x 15 mm x 5 mm 使い捨て型の埋め込みの蝸牛を完全にカバーする化合物。Whitlonらによる記述で埋め込み型の下に平行になるので、その側に蝸牛を向けます。14
</li>
	<li>すぐに OCT を固めるし、一晩-80 ° C で保存にドライアイスの型を置きます。</li>
	<li>0.01% の顕微鏡スライドを浸し室温 30 分削除でポリ L リジンのスライド、すすぎ、していない一晩乾燥することができます。凍結切片のこれらのスライドを使用します。</li>
	<li>-80 ° C のフリーザーから 10 月に蝸牛を削除し、ドライアイスの上に置きます。シャープのミクロトーム刃 (L x w: 80 mm × 8 mm、厚さ 0.25 mm、34 ° のカット角) を使用してセクション-30 ° C でクライオスタットを用いた 10 μ m で 10 月のブロックスライドごとの 2 つのセクションを配置します。</li>
	<li>スライド 4 ° c が終わったら冷蔵庫で冷やします。</li>
</ol>5. 免疫組織化学
<ol><li>スライド ラックの上に料理を染色ガラス顕微鏡スライドにスライドを配置します。350 mL の 1x PBS で皿を満たし、室温で 5 分間スライドを 3 回洗浄します。</li>
	<li>お皿からスライドを削除し、周辺組織の組織オフに乾燥しないように確認して、乾燥のワイプを使用してオフに乾燥します。<ol>
<li>液体ブロックのペンを使用して、組織切片周囲円を描画します。</li>
	</ol>
</li>
	<li>ブロッキング溶液 10% 通常 (ロバ) 血清, 1% 非イオン性洗剤および 1x PBS で 1% BSA 150 μ L を追加することによって部屋の温度で 1 時間のための組織をブロックします。</li>
	<li>ブロッキング液の過剰をオフにタップし、一晩で 4 ° C (ロバ) 10% 正常血清、0.1% 非イオン性洗剤、一次抗体 (下のメモを参照) の 150 μ L で加湿チャンバー組織をインキュベート 1x PBS で。 注:次抗体これらの希釈液は使用された:図 2および3、p STAT1 Ser727 1: 300 のため。図 4、p STAT1 Ser727、1: 100、1: 100 TRPV1 の</li>
	<li>スライド ラックの上に料理を染色ガラス顕微鏡スライドにスライドを配置します。350 mL の 1x PBS で皿を満たし、室温で 5 分間スライドを 3 回洗浄します。</li>
	<li>0.01% 非イオン性洗剤と暗闇の中で加湿チャンバーに室温で 2 に 3 h の 1x PBS で 10% 正常 (ロバ) 血清を含む溶液で希釈した二次抗体とインキュベートします。 注:次の二次抗体これらの希釈液は使用された: の図 2と3ロバ抗うさぎ IgG 1:600。図 4ローダミン (TRITC) ロバ抗うさぎ IgG 1: 500 とロバの反やぎ IgG 1: 500。</li>
	<li>スライド ラックの上に料理を染色ガラス顕微鏡スライドにスライドを配置します。350 mL の 1x PBS で皿を満たし、室温で 5 分間スライドを 3 回洗浄します。</li>
	<li>スライドの外の余分な液体をタップし、周囲の組織の乾燥のワイプを使用してスライドを乾燥します。組織に直接 DAPI で取り付けのエージェントの 1 つのドロップを追加することによって、スライドをマウントします。ゆっくり下に気泡を形成しないことを確保する上、coverslip の場所し、暗闇の中で室温で一晩治すためスライドを許可します。4 ° C でスライドを保存します。</li>
	<li>共焦点の顕微鏡を使用してスライドをイメージします。レーザーを使用して、次のように画像の: ロバ抗うさぎ IgG、488 nm レーザー DAPI ・ ローダミン TRITC 543 nm レーザー紫外線レーザー。</li>
</ol>

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利害の対立が宣言されていません。

トランス鼓膜投与経路重要な全身性副作用全身投与する場合それ以外の場合になる蝸牛に薬や他のエージェントのローカライズされた配信できます。この投与法は、全身の経路を通じて達成されるとより有意に高い用量でアクションのサイトに薬の迅速なアクセスをできます。結果ここまで提示して公開以前ことを示したトランス鼓膜 [R] - N - フェニル イソプロピル アデノシン (R</...

聴覚末梢系は、シスプラチン、アミノグリコシド系抗生物質などの薬に絶妙に敏感です。さまざまな固形腫瘍、卵巣、精巣、ヘッドなどの治療に広く使用されている抗癌剤は、シスプラチンと頸部の癌。この薬の使用を経験した難聴は線量制限と1を扱う患者の 75-100% に影響を与える非常に一般的。カルボプラチン オキサリプラチンなどの他の薬をシスプラチン2,3,4、5、選択肢として浮上しているが、その有用性は、いくつかの癌に限られています。
初期の研究では、シスプラチン、アミノグリコシド系抗生物質による難聴を媒介に活性酸素種 (ROS) の重要な役割を示しています。その後の研究を示し、NADPH オキシダーゼの NOX3 アイソ フォーム、蝸牛の ROS のプライマリ ソースであるシスプラチン6,7をアクティブに。ROS 妥協の世代抗酸化につながる細胞の緩衝能力は8細胞膜の脂質過酸化反応を増加しました。また、シスプラチンは非常に有毒アルデヒド 4-ヒドロキシノネナール (4 HNE)、細胞死9,10のイニシエーターを生成するヒドロキシルラジカルの生産を増加します。これらの調査結果に基づいて、いくつかの抗酸化物質はシスプラチン難聴の治療のために検討されています。N - アセチルシステイン (NAC)、チオ硫酸ナトリウム (STS) や amifostine、D-メチオニンが含まれます。しかし、抗酸化療法の主要な心配はこれらの抗酸化物質がシスプラチンの抗酸化剤分子のチオール基との相互作用を通じて11を全身投与した場合シスプラチン化学療法有効性を減らすことができます。
抗酸化療法とこれらの問題の観点から本研究の目的は聴力損失を減らすために蝸牛に酸化防止剤と他の薬を提供するトランス鼓膜のルートを検討しました。特に有望な薬物と短い干渉 (si)、RNA、後述のトランス鼓膜のルートが表示されます。

<table><tbody><tr><td>Ketathesia (100 mg/ml) 10 ml</td><td>Henry Schein</td><td>56344</td><td>Controlled substance&amp;nbsp;</td></tr><tr><td>AnaSed Injection/Xylazine (20 mg/ml) 20 ml</td><td>Henry Schein</td><td>33197</td><td></td></tr><tr><td>2.5 mm disposable ear specula</td><td>Welch Allyn</td><td>52432</td><td></td></tr><tr><td>Surgical Scope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>29 G X 1/2 insulin syringe</td><td>Fisher Scientific</td><td>14-841-32&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>cis-Diammineplatinum(II) dichloride</td><td>Sigma Aldrich</td><td>P4394</td><td>TOXIC - wear proper PPE</td></tr><tr><td>Harvard 50-7103 Homeothermic Blanket Control Unit</td><td>Harvard Apparatus</td><td>Series 863</td><td></td></tr><tr><td>Excel International 21 G X 3/4 butterfly needle</td><td>Fisher</td><td>14-840-34&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>BSP Single Speed Syringe Pump</td><td>Brain Tree Sci, Inc</td><td>BSP-99</td><td></td></tr><tr><td>Pulse Sound Measurement System</td><td>Bruel &amp;amp; Kjaer</td><td>Pulse 13 software</td><td></td></tr><tr><td>High-Frequency Module</td><td>Bruel &amp;amp; Kjaer</td><td>3560C</td><td></td></tr><tr><td>1/8&amp;Prime; Pressure-field Microphone &amp;mdash;-Type 4138</td><td>Bruel &amp;amp; Kjaer</td><td>bp2030</td><td></td></tr><tr><td>High Frequency Transducer</td><td>Intelligent Hearing System</td><td>M014600</td><td></td></tr><tr><td>Opti-Amp Power Transmitter</td><td>Intelligent Hearing System</td><td>M013010P</td><td></td></tr><tr><td>SmartEP ABR System</td><td>Intelligent Hearing System</td><td>M011110</td><td></td></tr><tr><td>Disposable Subdermal EEG Electrodes</td><td>CareFusion</td><td>019-409700</td><td></td></tr><tr><td>16% Formaldehyde, Methanol-free</td><td>Fisher Scientific</td><td>28908</td><td>TOXIC - wear proper PPE&amp;nbsp;</td></tr><tr><td>7 mL Borosilicate Glass Scintillation Vial</td><td>Fisher Scientific</td><td>03-337-26</td><td>Can be purchased through other vendors</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP118-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Tissue Plus OCT Compound</td><td>Fisher Scientific</td><td>4585</td><td></td></tr><tr><td>CryoMolds (15 mm x 15 mm x 5mm)</td><td>Fisher Scientific</td><td>22-363-553</td><td>Can be purchased through other vendors</td></tr><tr><td>Microscope Slides (25mm x 75mm)</td><td>MidSci</td><td>1354W</td><td>Can be purchased through other vendors</td></tr><tr><td>Coverslips (22 x 22 x 1)</td><td>Fisher Scientific</td><td>12-542-B</td><td>Can be purchased through other vendors</td></tr><tr><td>Poly-L-Lysine Solution (0.01%)</td><td>EMD Millipore</td><td>A-005-C</td><td>Can be purchased through other vendors</td></tr><tr><td>HM525 NX Cryostat</td><td>Thermo Fischer Scientific</td><td>956640</td><td></td></tr><tr><td>MX35 Premier Disposable Low-Profile Microtome Blades</td><td>Thermo Fischer Scientific</td><td>3052835</td><td></td></tr><tr><td>Wheaton&amp;trade;&amp;nbsp;Glass 20-Slide Staining Dish with Removable Rack</td><td>Fisher Scientific</td><td>08-812</td><td></td></tr><tr><td>Super Pap Pen Liquid Blocker</td><td>Ted Pella, Inc.</td><td>22309</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-121</td><td>Can be purchased through other vendors</td></tr><tr><td>TritonX-100</td><td>Acros</td><td>21568</td><td>Can be purchased through other vendors</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>Can be purchased through other vendors</td></tr><tr><td>Phospho-Stat1 (Ser727) antibody</td><td>Cell Signaling</td><td>9177</td><td></td></tr><tr><td>VR1 Antibody (C-15)</td><td>Santa Cruz</td><td>sc-12503</td><td></td></tr><tr><td>DyLight 488 Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-485-152</td><td>Discontinued</td></tr><tr><td>DyLight 488 Donkey anti Goat</td><td>Jackson Immuno Research</td><td>705-485-003</td><td>Discontinued</td></tr><tr><td>Rhodamine (TRTIC) Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-025-152</td><td>Discontinued</td></tr><tr><td>ProLong&amp;reg; Diamond Antifade Mountant w/ DAPI</td><td>Thermo Fisher</td><td>P36971</td><td></td></tr><tr><td>(&amp;minus;)-N6-(2-Phenylisopropyl)adenosine</td><td>Sigma Aldrich</td><td>P4532</td><td></td></tr><tr><td>8-Cyclopentyl-1,3-dipropylxanthine</td><td>Sigma Aldrich</td><td>C101</td><td></td></tr><tr><td>siRNA pSTAT1</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>siRNA NOX3</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Scrambled Negative Control siRNA</td><td>Qiagen</td><td>1022076</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr></tbody></table>

trans-tympanic drug delivery for the treatment of ototoxicity

薬剤性難聴を治療するために保護剤の全身投与は、これらの保護剤が主な薬の化学療法の有効性と干渉する可能性可能性によって制限されます。これはその抗癌アクションは聴力損失に対して十分な保護を提供する抗酸化物質によって減衰は薬シスプラチンの特に当てはまります。他の現在か潜在的な otoprotective のエージェントは、全身投与する場合、同様の問題を引き起こす恐れが。限られた全身の副作用とローカルでこれらのエージェントの高いレベルの蝸牛に直接さまざまな生物学的製剤または保護剤のアプリケーションになります。本報告では、我々 は、蝸牛蝸牛に関する基礎科学研究を進めようと診療所で otoprotective エージェントの使用を指示する簡単な方法を提供するさまざまな薬や生物学的試薬の配信のトランス鼓膜メソッドを示します。このレポートは、トランス鼓膜薬物送達法の詳細をどのようにこの技術が正常に実験動物の治療に使用されてシスプラチン難聴の例を示します。

ABR 反応閾値の有意な上昇を示した次のシスプラチン投与 3 日間ラットを用いて測定します。トランス鼓膜 [R] - N - phenylisopropyladenosine 投与ラットにおけるこれらのしきい値の上昇を有意に減少した (R-ぴあ)、アデノシン1受容体アゴニスト15シスプラチンの前に。Rの作用の特異性-1受容体は、それが 8-シクロペンチル...

Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

蝸牛にトランス鼓膜のルートを通じて医薬品の集中管理のための手法を提案します。この経路によるドラッグデリバリー シスプラチン化学療法剤の抗腫瘍効果と干渉しません。

難聴の治療のためのトランス鼓膜ドラッグデリバリー

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

trans-tympanic-drug-delivery-for-the-treatment-of-ototoxicity

Research

JoVE Journal

Medicine

9.3K ビュー.  Southern Illinois University School of Medicine. 蝸牛にトランス鼓膜のルートを通じて医薬品の集中管理のための手法を提案します。この経路によるドラッグデリバリー シスプラチン化学療法剤の抗腫瘍効果と干渉しません。

ビデオ: Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

腹部腫瘍の治療のための熱的アブレーション

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

医学

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

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

聴覚神経の光遺伝学的刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

神経科学

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

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

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 ...

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 ...

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 ...

Endaural Endoscopic Atticoantrotomy (Retrograde Mastoidectomy) using a Constant Suction Bone-drilling Technique

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the main technical challenge of endoscopic middle ear surgery. The accompanying video describes the detailed protocol of a constant-suction bone-drilling technique and the procedure of endaural exclusive endoscopic atticoantrotomy (retrograde mastoidectomy) using this technique. The main components of this bone-drilling technique include a soft and flexible suction tube, which is placed into the tympanic cavity to provide constant suction, and a soft sleeve, which is wrapped around the drill shaft to prevent the high-speed rotating shaft from damaging the lens of the endoscope. With these simple modifications, the traditional otological electrodrill can be used for drilling a tiny endaural incision in endoscopic middle ear surgery. Based on this bone-drilling technique, endaural endoscopic atticoantrotomy (retrograde mastoidectomy) can be successfully established for the removal of various amounts of bone, depending on the extent of the lesion. The short-term postoperative outcome seems promising.

Endaural Endoscopic Atticoantrotomy Retrograde Mastoidectomy using a Constant Suction Bone-drilling Technique

In this paper, we present the protocol and short-term outcomes of endaural exclusive endoscopic atticoantrotomy using a constant suction bone-drilling technique.

内耳内視鏡的付着瘻切除術(逆行性乳房切除術)は、一定の吸引骨穿孔技術を使用して

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the ...

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. ...

生物学

<meta charset="utf8"></head><body>電気音響刺激における長電極による聴力保存

Enhanced Cochlear Coverage and Hearing Preservation in High-Frequency Hearing Loss via Electric Acoustic Stimulation with Longer Electrode

Electric-acoustic stimulation (EAS) is a promising treatment to improve hearing ability in patients with high-frequency hearing loss (HL). In EAS surgeries, shorter electrodes have been preferred to avoid the presence of an electrode covering the residual hearing region. However, our earlier studies showed that EAS with longer electrodes (28 mm) could preserve acoustic hearing. Additionally, we reported that the hearing preservation (HP) scores were independent of the length of the inserted electrodes, consistent with the systematic review. As most EAS patients gradually lose residual hearing over time due to the natural course of HL, in these cases, providing broader cochlear coverage using longer electrodes was beneficial toward better place-pitch matching. In addition to preparing for the deterioration in&#160;hearing in&#160;the future, EAS with longer electrodes could offer various types of map strategies. Herein, we show the pre-, intra-, and post-procedures for EAS surgery. Appropriate preoperative evaluation, less invasive surgery, flexible lateral-wall electrodes, and steroid administration resulted in good HP following EAS with longer electrodes.

<meta charset="utf8"></head><body>著者スポットライト:蝸牛のカバー範囲と聴力保存を強化するための長い電極によるEASの最適化

Electric acoustic stimulation (EAS) with longer electrodes can offer broader cochlear coverage and various types of maps in cases of high-frequency hearing loss. Combining less invasive surgery, flexible lateral-wall electrodes, and steroid administration permits deeper insertion with little or no surgical trauma, resulting in good preservation of hearing.