Name: canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice
Uploaded: 2018-05-25T13:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice at JoVE.com

この作品は、中国の国家自然科学基金 (許可番号 81570912、81771016、81100717) によって支えられました。

動物愛護と中国の首都医科大学の利用委員会のガイドラインによるとすべてのプロシージャと動物の手術を行った。
1. デバイスの準備
<ol><li>注入カニューレ (図 1 a) をするためには、ポリイミド チューブを接続 (内径 114.3 μ m、外径 139.7 μ m、長さ 3 ~ cm) ポリエチレン チューブに (内径 280 μ m、外径 640 μ m、長さ ~ 40 cm)。瞬間接着剤を使用して、少なくとも 3 つのアプリケーションとの接続をシールします。酸化エチレンと注入カニューレを消毒します。 注: 管を密閉する際は、ポリイミド チューブ、カニューレの閉塞になることができますを入力してから瞬間接着剤を防止します。ガス滅菌は、接続時の漏洩を引き起こす可能性があります少なくとも 3 回でチューブをシールします。</li>
	<li>30 G の針を使用して、食塩を含む 1 cc シリンジにポリエチレン管の端を接続します。通常生理食塩水で漏洩またはポリエチレン チューブ、ポリイミド チューブと接続で閉塞をチェックする 1 cc のシリンジでの注入によるカニューレを埋めます。 注: 漏れや接続の閉塞、カニューレが次の手順で使用できません。</li>
	<li>通常生理食塩水で 10 μ L のマイクロ注射器を避難し、注入カニューレと前述の 30 G 針に接続します。インジェクション ポンプ (図 1 b) にマイクロ シリンジをインストールします。射出速度を 0.5 μ L/分と高速グリーン色素と AAV8-GFP、1 μ L にボリュームまたはストレプトマイシンの 2 μ L を設定します。 注: 射出速度の推奨範囲は 0.1−0.5 μ L/分、および注入量の推奨範囲は 0.5−2 μ L。</li>
	<li>1 μ L にマイクロ シリンジを引き戻すし、試薬の注入を抽出します。通常の生理食塩水と注射剤 (図 1) の間に空気の隙間が形成されます。</li>
	<li>必要に応じてマーカーペンを使ってポリエチレン チューブのガス-液境界線の位置にラベルを付けます。これは、注入中に試薬の流れを監視する使用ことができます。</li>
</ol>2。 成体マウスの Canalostomy
<ol><li>成体マウスの麻酔 (女性、FVB/N、5、6 週齢) ケタミン塩酸 (120 mg/kg 腹腔内投与による、キシラジン塩酸 (7 mg/kg)。動物が痛みを伴う刺激 (つま先ピンチ反射) への応答を示さないまで 5−10 分間待ちます。麻酔後、予熱した電気パッドに動物を配置します。メロキシカム (1 mg/kg)、鎮痛薬は、手術の前に皮下に適用されました。</li>
	<li>眼軟膏と動物の目をカバーします。電気動物クリッパーと左後耳介領域を剃るし、75% エタノールで 3 回皮膚を消毒します。</li>
	<li>予熱した電気パッドに動物を配置します。~ 37 ° c. に電気パッドの温度を設定します。左耳の手術を容易にするために右側臥位に動物を配置します。</li>
	<li>1 1.5 cm 後耳介切開 〜 3 mm 左着けた溝 (図 2 a) を作る。</li>
	<li>位置決め (PSC) 後半規管、外側半規管 (LSC): PSC と LSC、通常 9 に 3 として原点と、calvarium に平行な平面として耳介 (図 2 bで大胆なドット) のルートを定義するとき〜 3 mm 2 と 3 (図 2 b) 耳介のルートをあります。</li>
	<li>にべもなく PSC と余白がはっきりと側頭骨 (図 2) の暗い縞として見える LSC を公開するマイクロ鉗子で頭骨を覆う筋肉を解剖します。LSC では、calvarium に平行な平面から角度を約 30 ° と PSC は、LSC (図 2) に垂直。マイクロ鉗子で筋肉の小片を収集し、乾燥させてください。次の手順でこの筋肉が穴をシールに使用されます。 注: は、筋肉への損傷を最小限にするために組織の過剰なテアリングを回避します。PSC (図 2 D) を公開するとき隣接する船の損傷を防ぐ。</li>
	<li>26 G 針 (図 2 D) を使用して PSC の真ん中の部分に小さな穴を作る。穴から液漏れは、PSC の骨の壁の成功浸透を示します。適切なサイズ、ポリイミド チューブの直径よりわずかに大きい穴を拡大します。 注: ドリルし、優しく、徐々 に穴を拡大、PSC の破壊を避けるために。</li>
	<li>胸水綿ペレットを使用して PSC の穴の周囲をクリーンアップします。</li>
	<li>1−2 mm (図 2 e) の深さに cru コミューンに向かって PSC に優しくポリイミド チューブの先端を挿入します。ポンプの '実行' ボタンを押して注射を開始します。</li>
	<li>注入後の拡散試薬 〜 2 分を待ちます。マイクロはさみで 2.6 ステップで収集され筋肉の小さな部分をトリミングします。次に、注入カニューレを削除し、すぐに PSC の穴に筋肉を配置します。 注: は、穴が完全に密封されていることを確認する接続した後穴から漏れる液体を確認します。</li>
	<li>区切られた筋肉と皮下組織を返します。5-0 縫合糸を使用して切開部を縫合します。ポビドン ヨードと切開の地域を消毒します。 注: 上記すべての手術は、約 25 分かかります。</li>
	<li>~ 37 ° C に予熱した電気パッド上に動物を配置します。右側臥位の回復のために動物を配置します。</li>
	<li>聴性脳幹反応 (ABR) の測定を行い、水泳のテスト18手術後 1 週間。</li>
	<li>必要な場合は、PSC や LSC の別の領域に繰り返し注射を実行します。</li>
</ol>3. 新生児マウス Canalostomy
<ol><li>低体温症を誘導し、生後 1、2 (P1-2) で新生児マウス (FVB/N) で鎮静を維持に使用してください。~ 4 分の砕いた氷のプラスチックのラップで覆われたベッドの上には、子犬を配置します。次に、氷で満たされたプラットフォームで子犬を置きます。75% エタノールで 3 回手術野を消毒します。 注: は、子犬の頭が氷のことを確認します。氷で満たされたプラットフォームで子犬と全体の手術を行います。</li>
	<li>次の手順で異なる大人のマウスと比較して新生児の。<ol>
<li>耳介の折り目に後方から ~ 2 mm 〜 3 mm 耳介後部切開を作る (図 3 a -B)。</li>
		<li>優しく 26 G 針を使用してソフトの PSC に開口部を作る。開口部を拡大せずに、PSC にカニューレを挿入 (図 3 -E)。</li>
		<li>注入後、ソフト PSC (図 3 f) の破壊につながることができます、後者として、開口部を接続するのではなく、カバー、筋の部分を使用します。</li>
		<li>6-0 縫合糸を使って皮膚を閉じます。</li>
		<li>ABR の測定を行い、P3030水泳のテスト。</li>
	</ol>
</li>
	<li>親の共食いは新生児手術後一般的な問題です。次の手順は、親の共食いの可能性を減らします。<ol>
<li>周囲のアルコール綿を使用して切開手術後血をクリーンアップします。</li>
		<li>新生児がダムに戻る前に自由に移動できることを確認します。</li>
		<li>新生児を母親のケージと新生児がゴミの真ん中に戻って配置から汚れた寝具に汚します。</li>
		<li>似たような耳介後部切開や縫合手術を受けるしないいずれかが表示されます。</li>
		<li>飼育ケージから男性を分離します。</li>
	</ol>
</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmastoid+semicircular+canal+occlusion:+a+safe+and+highly+effective+treatment+for+benign+paroxysmal+positional+vertigo+and+superior+canal+dehiscence.">Transmastoid semicircular canal occlusion: a safe and highly effective treatment for benign paroxysmal positional vertigo and superior canal dehiscence.</a> Laryngoscope. 122 (8), 1862-1866 (2012).</li><li>Naples, J. G., Eisen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+and+Evolution+of+Surgery+on+the+Vestibular+Labyrinth.">The History and Evolution of Surgery on the Vestibular Labyrinth.</a> Otolaryngol Head Neck Surg. 155 (5), 816-819 (2016).</li><li>Hamilton, L., Keh, S., Spielmann, P. M., Hussain, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+we+do+it:+locating+the+posterior+semicircular+canal+in+occlusion+surgery+for+refractory+benign+paroxysmal+positional+vertigo:+a+cadaveric+temporal+bone+study.">How we do it: locating the posterior semicircular canal in occlusion surgery for refractory benign paroxysmal positional vertigo: a cadaveric temporal bone study.</a> Clinical Otolaryngology. 41 (2), 190-193 (2016).</li><li>Jung, J. 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=siRNA+targeting+Hes5+augments+hair+cell+regeneration+in+aminoglycoside-damaged+mouse+utricle.">siRNA targeting Hes5 augments hair cell regeneration in aminoglycoside-damaged mouse utricle.</a> Mol Ther. 21 (4), 834-841 (2013).</li><li>Kamiya, 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=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> Am J Pathol. 171 (1), 214-226 (2007).</li><li>Pfannenstiel, S. C., Praetorius, M., Plinkert, P. K., Brough, D. E., 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=Bcl-2+gene+therapy+prevents+aminoglycoside-induced+degeneration+of+auditory+and+vestibular+hair+cells.">Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.</a> Audiol Neurootol. 14 (4), 254-266 (2009).</li><li>Kawamoto, K., Izumikawa, M., Beyer, L. A., Atkin, G. 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=Spontaneous+hair+cell+regeneration+in+the+mouse+utricle+following+gentamicin+ototoxicity.">Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.</a> Hear Res. 247 (1), 17-26 (2009).</li><li>Wang, G. P., 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=Notch+signaling+and+Atoh1+expression+during+hair+cell+regeneration+in+the+mouse+utricle.">Notch signaling and Atoh1 expression during hair cell regeneration in the mouse utricle.</a> Hear Res. 267 (1-2), 61-70 (2010).</li><li>Pietola, L., 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=HOX-GFP+and+WOX-GFP+lentivirus+vectors+for+inner+ear+gene+transfer.">HOX-GFP and WOX-GFP lentivirus vectors for inner ear gene transfer.</a> Acta Otolaryngol. 128 (6), 613-620 (2008).</li><li>Han, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li></ol>

利害の対立が宣言されていません。

本研究で我々 は canalostomy による薬物が蝸牛と前庭エンドの臓器全体で試薬の広範な分布の結果を示した。内耳遺伝子配達方法として canalostomy は、聴覚と前庭の機能に最小限のダメージで成人と新生児マウスの内側の耳の GFP 発現をもたらした。さらに、同じ動物の複数の注射を簡単に実行できます。
Canalostomy の最大の強みの 1 つは、成体マウス16,<...

難聴と前庭機能障害に影響を与える患者の相当な数を密接に感音難聴内耳障害に関連付けられています。内耳への治療薬の配達は、内耳障害の治療のための約束を示しています。全身またはローカル アプローチは、内耳に薬を提供する使用できます。いくつかの内耳疾患は、突発性難聴、全身ステロイド1と扱われる一般的などの全身性の薬物投与による正常に扱われます。さらに、レンツらはアンチセンス オリゴヌクレオチドの全身投与は難聴を改善し、バランスの Ush1c 突然変異体マウス モデル2で機能することを示した。ただし、内耳疾患の大部分が効果的に扱われません全身投与による内耳の3,4薬へのアクセスを制限する血液迷路障壁のため。対照的に、局所薬物配送戦略は内耳障害をより効率的に扱うことができます。確かに、内耳は潜在的薬剤の局所投与; の理想的なターゲット1 サイトの拡散や注射後薬の普及を促進する液体でみたされている、それは比較的孤立した隣接臓器から副作用5,6を制限します。
局所薬物配送戦略鼓と intralabyrinthine のメソッドがあります。鼓の経路の有効性は大きく薬物透過 RWM3,4,7,8薬の滞留時間と丸い窓膜 (RWM) に依存します。したがって、薬物や、RWM を突き通すことができない試薬の配信に適したはありません。Intralabyrinthine 方法は、薬の高用量との広範な分布の結果、内耳に直接接種を含みます。ただし、intralabyrinthine 方法は繊細な手術を必要とし、侵略的、内耳機能へのダメージに 。現在、人間9で使用するため十分に安全であることは実証されていないと、intralabyrinthine 注射薬は動物実験でのみ使用されます。したがって、手術の手順を簡略化する必要がありますと怪我のリスク低減、クリニックに intralabyrinthine アプローチを翻訳します。
RWM5,10、11 scala メディア12,13,14スカラー座索注入による動物のいくつかの intralabyrinthine アプローチが評価され15,16、スカラー座附17、18,16,2019,半規管、および内リンパ嚢21。それぞれのアプローチには長所と短所6。RWM による配信は、新生児マウス5,22の非外科的です。ただし、軽度の聴力損失は RWM 注入23、中耳手術24後胸水のため後成体マウスで観察されます。感覚上皮を含む内の空間に直接試薬の注入が含まれます、Scala 媒体注入ターゲット エンド臓器12,14,の高い試薬濃度を実現します。25,26。 このアプローチ複雑な手順が必要ですし、生後 5 (P5) よりも後に実行された場合、聴覚閾値の有意な上昇の結果25,は27、そのアプリケーションを制限します。
Canalostomy は特に成体マウス16,18,28,29,30である内側の耳に与えるダメージを最小限を引き起こす intralabyrinthine の上記のアプローチと比較して、保護効果と並進の側面の評価にとって重要です。さらに、齧歯類では半規管は肺嚢胞手術を容易にし、手術中に中耳の障害を回避する以外にあります。クリニック、半規管の手術では、難治性良性発作性頭位めまい症31,32,33, canalostomy の臨床的可能性を示唆しているが使用されます。以来、まず、河本らによって記述されていた。マウス内耳18,19,28,29 にウイルスのベクトル、siRNA、幹細胞は、アミノグリコシド系などの様々 な試薬を提供する16年では、canalostomy が使用されています ,34,35,36,37。Canalostomy によるアデノ随伴ウイルス (AAV) ベクターの接種感覚上皮における外来遺伝子の過剰発現と蝸牛と前庭エンドの臓器18,28,の一次ニューロンの特性を有効にする29,30。Canalostomy による遺伝子治療を有バランス機能を復元し、人間アッシャー症候群19、その canalostomy が cochleovestibular の遺伝的疾患に対する遺伝子治療の研究に有用のマウスモデルで聴覚を向上させます。Canalostomy による間葉系幹細胞の移植は、蝸牛線維細胞と急性の感音難聴の聴力損失35のラットモデルにおける聴力回復の再編成の結果します。また、前庭神経病変18,34,38, を確立する内耳にアミノグリコシド系抗生物質を導入する canalostomy を使用できます、18を必要な場合、複数の注射を実行できます。,34。
本稿で述べる、成人および新生児マウスにおける canalostomy 技術。緑色蛍光タンパク質 (GFP) 遺伝子 (AAV8-GFP) とストレプトマイシン、高速グリーン色素と AAV 血清型 8 (AAV8) などを含むさまざまな試薬を接種したマウス内耳 canalostomy 後即時および長期的な成果を評価するために。

<table><tbody><tr><td>Polymide Tubing</td><td>A-M Systems</td><td>823400</td><td></td></tr><tr><td>Polyethylene Tubing</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/1</td><td></td></tr><tr><td>10&amp;mu;l Microsyringe</td><td>Hamilton Company</td><td>80001</td><td></td></tr><tr><td>Xylazine HCL</td><td>Sigma-Aldrich Co. Llc.</td><td>X-1251</td><td></td></tr><tr><td>Operating Miroscope</td><td>Carl Zeiss Optical LLC.</td><td>Pico</td><td></td></tr><tr><td>Micro Forceps</td><td>Dumont Dumostar</td><td>10576</td><td></td></tr><tr><td>Fast-green Dye</td><td>Sigma-Aldrich Co. Llc.</td><td>F7252</td><td></td></tr><tr><td>AAV8-GFP</td><td>BioMiao Biological Technology Co. Ltd (Beijing, China)</td><td>20161101</td><td>Titer: 2&amp;times;10e12 vg/mL</td></tr><tr><td>Streptomycin Sulfate</td><td>Sigma-Aldrich Co. Llc.</td><td>S9137</td><td></td></tr><tr><td>Microinjection Pump</td><td>Stoelting Co.</td><td>789100S</td><td></td></tr><tr><td>Electric Pad</td><td>Pet Fun</td><td>11072931136</td><td></td></tr><tr><td>1 cc Syringe</td><td>Mishawa Medical Industries Ltd. (Shanghai, China)</td><td>2011-3151258</td><td></td></tr><tr><td>Ketamine HCL</td><td>Gutian Pharmaceutical Co., Ltd. (Fujian, China)</td><td>H35020148</td><td></td></tr><tr><td>Electric Animal Clipper</td><td>Codos Electrical Appliances Co., Ltd. (Guangdong, China)</td><td>CP-8000</td><td></td></tr><tr><td>Cotton Pellet</td><td>Yatai Healthcare Ltd. (Henan, China)</td><td>Yu-2008-1640081</td><td></td></tr><tr><td>Suture</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd. (Shanghai, China)</td><td>Hu-2013-2650207</td><td></td></tr><tr><td>Eye Ointment</td><td>Beijing Shuangji Pharmaceutical Ltd. (Beijng China)</td><td>H11021270</td><td></td></tr></tbody></table>

canalostomy as a surgical approach to local drug delivery into the inner ears of adult and neonatal mice

内耳に治療薬の局所投与は、内耳疾患に対する有望な治療法です。半規管 (canalostomy) を注入は、内耳への薬剤の局所投与に有用なアプローチをする示されています。この記事の目的は、について、詳しく説明、成人と新生児の両方のマウスの canalostomy に関与する手技です。蝸牛と前庭のエンドの聴覚に損傷が少なく臓器で注入された試薬の広範な分布を容易に、canalostomy として高速グリーン色素と緑色蛍光タンパク質遺伝子をアデノ随伴ウイルス血清型 8 によって示されると前庭の機能。成人および新生児マウス; 手術が実施されました確かに、必要な場合は、複数の手術を実行でした。結論としては、canalostomy は成人および新生児マウス内耳に薬物送達への効果的かつ安全なアプローチで、将来的に人間の内耳疾患を治療に使用することがあります。

高速グリーンの色素は、成人および新生児マウス内耳における即時分布を評価するための PSC に注入されました。手術 (図 4) 直後後、蝸牛、前庭、半規管の中で染料が検出されました。
安全性と内耳遺伝子送達用 canalostomy の効率を評価するには、AAV8 GFP は成人および新生児マウス内耳に注入されま?...

Watch this Scientific Journal Video about Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice at JoVE.com

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

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

ここで我々 は聴覚と前庭の機能に最小限のダメージで半規管を介して成人および新生児マウスの内耳への薬剤の局所投与を可能にする canalostomy の手順をについて説明します。このメソッドは、マウス内耳へのウイルスのベクトル、医薬・小分子を接種する使用ことができます。

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

canalostomy-as-surgical-approach-to-local-drug-delivery-into-inner

Research

JoVE Journal

Genetics

10.6K ビュー.  Capital Medical University. ここで我々 は聴覚と前庭の機能に最小限のダメージで半規管を介して成人および新生児マウスの内耳への薬剤の局所投与を可能にする canalostomy の手順をについて説明します。このメソッドは、マウス内耳へのウイルスのベクトル、医薬・小分子を接種する使用ことができます。

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

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

in vitro転写アッセイおよび創薬への応用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

遺伝学

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

複数の蛍光によるインター染色体安定型異常の検出<em&gt;その場で</em&gt;ハイブリダイゼーション（mFISH）と照射されたマウスにおけるスペクトル核型分析（SKY）

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

誘導性一過性トランスフェクションを介して制御可能なイオンチャネルの発現

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

マウスの脳に直接薬剤送達のための異粘膜移植手順

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

医学

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 ...

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 ...

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. ...

生物学

Canalostomy for Local Drug Delivery into the Inner Ear of an Adult Mouse

Source: Guo, J. et al., <a href="https://app.jove.com/v/57351">Canalostomy as a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice.</a>&#160;J. Vis. Exp. (2018)
This video showcases canalostomy, a procedure that allows local drug delivery into the inner ear of a mouse through the semicircular canal with minimal damage to hearing and vestibular function. This method enables the inoculation of viral vectors, pharmaceuticals, and small molecules into the mouse&#39;s inner ear.

成体マウスの内耳への局所薬物送達のためのカナロストミー

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Device ...

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

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