Name: surgical labyrinthectomy of the rat to study the vestibular system
Uploaded: 2018-05-19T13:00:00.000+00:00
Duration: 4 min 3 s
Description: Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

本研究は韓国健康技術 R & D プロジェクトを通じて、韓国保健産業開発研究所 (KHIDI)、韓国の福祉・厚生省によって資金を供給からの助成金によって支えられた (許可番号: HI15C2651)。

本研究は, 機関動物ケアおよび使用委員会のソウル国立大学病院 (14-0148-C1A1) 評価・実験動物ケアの認定会の認定を受けているに従って国際。
注: 7-8 週古い (200/250 g) の Sprague-dawley (SD) ラットに実験を行った。各動物は、実験開始前 1 週間の実験室の条件に順応だった。定数 12 に温度と湿度管理された部屋で収容された動物-h:12-h: 明暗サイクル無料アクセスと食料と水。
1。 なるほど
<ol><li>オートクレーブの手術器具滅菌パッドをカバーします。70% のエタノールと営業領域を消毒します。滅菌ドレープを使用し、手術中に滅菌状態を維持します。</li>
	<li>ガウンを着用、マスク (カバー鼻と口)、キャップ (頭をカバー) と滅菌手袋のペア。手術器具は、滅菌の表面と接触して来る必要がありますのみ。</li>
	<li>チレタミン チレタミン麻酔薬 (40 mg/kg 体重) を注入または腹腔、大腿内側に筋肉とキシラジン (10 mg/kg 体重) 筋肉内あるいは腹腔。 注: 含むイソフルレン吸入麻酔の他のモードを使用もできます。</li>
	<li>(当初は 42 ° C で設定) 地球温暖化パッドにラットを配置します。麻酔下で目の乾燥を防ぐためにラットの両目に潤滑剤の眼軟膏を適用されます。</li>
	<li>ラットを右側を下に配置します。外科領域で毛皮をバリカンで剃る。70% のエタノールを使用してサイトを消毒します。</li>
	<li>左着けたエリアに 1% リドカイン塩酸塩を皮下注入します。</li>
	<li>#15 のメス刃を使えば、~5.0 cm 着けた切開。独立した筋肉と筋膜虹彩ハサミ (図 1 a) 外耳道を公開します。#15 ブレードまたはアイリスはさみ (図 1 b) を使用して、外耳道を少し開きます。鼓膜 (図 2 a) を広く公開します。</li>
	<li>顕微鏡 (7.5 X 12.5 X)、鼓膜と耳小骨鉗子を用いたアブミ骨を除いてを削除します。#15 ブレードやアイリス ハサミ (図 2 b) で lambdoidal の尾根筋をデタッチします。</li>
	<li>低速でドリルを使用して (< 3000 rpm) ダイヤモンドバー (2.1 または 1.4 mm)、(図 3 a) 顔面神経の出口のポイントのまわりの鼓室胞骨をドリルします。アブミ骨動脈を特定します。掘削のプロセス中にアブミ骨動脈を損傷しないように注意してください。</li>
	<li>アブミ骨動脈に優れた玄関を掘削を続行し、丸い窓と骨の外側半規管を公開します。外側半規管の膨大部付近を開きます。外側半規管の面にドリルを続行し、優れた半規管の膨大部をドリルします。</li>
	<li>18 G、22 G の針を使用して玄関の内容を吸引します。注入し、100% エタノール 3 の 1 cm3を吸い出しなさい x。</li>
	<li>筋肉と単純な縫合の二層で皮膚を閉じます。</li>
	<li>放置しないでください動物までそれは胸骨の横臥を維持するために十分な意識を取り戻しました。それは、加熱パッドまたは熱ランプを使用してキッスまでラットを暖かく保ちます。後に目覚め、ラットを客室に通常の環境条件で個々 のケージに移動します。</li>
	<li>抗生物質の投与 (例えば、セファドロキシル 20 mg/kg、トリメトプリム-スルホンアミド 100 μ L 皮下、またはペニシリン 300,000 IU); 1 日必要に応じて鎮痛薬 (オピオイドではない;例えば、Metacam カルプロフェン 2 mg/kg 5 mg/kg 皮下) 指定もできます。</li>
</ol>2. 偽手術
<ol><li>コントロールとして偽手術、(セクション 1) に従って同じ手術と手術後の手順を実行、外耳道と鼓膜 (ステップ 1.7)、鼓膜と耳小骨、除去の露出など、lambdoidal で筋肉の剥離リッジ (ステップ 1.8) が半規管を開くかエタノールを注入します。</li>
</ol>前庭機能の損失を確認します。
注: 前庭機能の喪失は、どちらかの行動または前庭機能検査を使用して評価できます。13,17,18 には行動テストには、眼振と姿勢の非対称性の評価が含まれます。
<ol><li>自発性または誘発バレル圧延、左側面に落ちたり18破壊側に傾いている間移動を含む姿勢の非対称性を識別します。ビデオ 1を参照してください。</li>
	<li>随意眼球運動の検査、眼振を識別します。自発眼振はいないとき休息状態の動物18頭優しく空気のパフします。ビデオ 2を参照してください。</li>
</ol>

<ol>
	<li>Curthoys, I. S., Halmagyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation:+a+review+of+the+oculomotor,+neural,+and+clinical+consequences+of+unilateral+vestibular+loss.">Vestibular compensation: a review of the oculomotor, neural, and clinical consequences of unilateral vestibular loss.</a> Journal of Vestibular Research. 5 (2), 67-107 (1995).</li><li>Smith, P. F., Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+recovery+following+unilateral+labyrinthectomy:+a+review.">Mechanisms of recovery following unilateral labyrinthectomy: a review.</a> Brain Research. Brain Research Reviews. 14 (2), 155-180 (1989).</li><li>Lacour, M., Helmchen, C., Vidal, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation:+the+neuro-otologist's+best+friend.">Vestibular compensation: the neuro-otologist's best friend.</a> Journal of Neurology. 263, S54-S64 (2016).</li><li>Darlington, C. L., Smith, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+recovery+from+vestibular+damage+in+mammals:+recent+advances.">Molecular mechanisms of recovery from vestibular damage in mammals: recent advances.</a> Progress in Neurobiology. 62 (3), 313-325 (2000).</li><li>Shinder, M. E., Ramanathan, M., Kaufman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+gene+expression+in+the+brain+during+acute+compensation+to+unilateral+vestibular+labyrinthectomy+in+the+Mongolian+gerbil.">Asymmetric gene expression in the brain during acute compensation to unilateral vestibular labyrinthectomy in the Mongolian gerbil.</a> Journal of Vestibular Research. 16 (4-5), 147-169 (2006).</li><li>Dutheil, S., Brezun, J. M., Leonard, J., Lacour, M., Tighilet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+and+astrogenesis+contribution+to+recovery+of+vestibular+functions+in+the+adult+cat+following+unilateral+vestibular+neurectomy:+cellular+and+behavioral+evidence.">Neurogenesis and astrogenesis contribution to recovery of vestibular functions in the adult cat following unilateral vestibular neurectomy: cellular and behavioral evidence.</a> Neuroscience. 164 (4), 1444-1456 (2009).</li><li>Gunther, 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=N-acetyl-L-leucine+accelerates+vestibular+compensation+after+unilateral+labyrinthectomy+by+action+in+the+cerebellum+and+thalamus.">N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.</a> PLoS One. 10 (3), e0122015 (2015).</li><li>Pericat, D., Farina, A., Agavnian-Couquiaud, E., Chabbert, C., Tighilet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+and+irreversible+unilateral+vestibular+loss:+a+novel+rat+model+of+vestibular+pathology.">Complete and irreversible unilateral vestibular loss: a novel rat model of vestibular pathology.</a> Journal of Neuroscience Methods. 283, 83-91 (2017).</li><li>Cass, S. P., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation+after+labyrinthectomy+and+vestibular+neurectomy+in+cats.">Vestibular compensation after labyrinthectomy and vestibular neurectomy in cats.</a> Otolaryngology-Head and Neck Surgery. 104 (1), 14-19 (1991).</li><li>Vignaux, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+chemical+model+of+vestibular+lesions+induced+by+arsanilate+in+rats.">Evaluation of the chemical model of vestibular lesions induced by arsanilate in rats.</a> Toxicology and Applied Pharmacology. 258 (1), 61-71 (2012).</li><li>Berryhill, W. E., Graham, 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=Chemical+and+physical+labyrinthectomy+for+Meniere's+disease.">Chemical and physical labyrinthectomy for Meniere's disease.</a> Otolaryngologic Clinics of North America. 35 (3), 675-682 (2002).</li><li>Morgenstern, C., Mori, N., Arnold, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+on+the+effect+of+labyrinth+anesthesia.">Experimental studies on the effect of labyrinth anesthesia.</a> Archives of Oto-Rhino-Laryngology. 237 (3), 255-261 (1983).</li><li>Nadasy, G. L., Raffai, G., Feher, E., Schaming, G., Monos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+standard+technique+for+labyrinthectomy+in+the+rat:+a+methodical+communication+with+a+detailed+description+of+the+surgical+process.">A simple standard technique for labyrinthectomy in the rat: a methodical communication with a detailed description of the surgical process.</a> Physiology International. 103 (3), 354-360 (2016).</li><li>Hitier, M., Besnard, S., Vignaux, G., Denise, P., Moreau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventrolateral+surgical+approach+to+labyrinthectomy+in+rats:+anatomical+description+and+clinical+consequences.">The ventrolateral surgical approach to labyrinthectomy in rats: anatomical description and clinical consequences.</a> Surgical and Radiologic Anatomy. 32 (9), 835-842 (2010).</li><li>Flourens, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recherches experimentales sur les propri&#233;t&#233;s et les fonctions du syst&#232;me nerveux dans les animaux vert&#233;br&#233;s. , (1824).</li><li>T'Ang, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+unilateral+labyrinthectomy+in+the+albino+rat.">The effects of unilateral labyrinthectomy in the albino rat.</a> Chinese Journal of Physiology. 10, 571-598 (1936).</li><li>Chang, M. 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=MicroRNAs+218a-5p,+219a-5p,+and+221-3p+regulate+vestibular+compensation.">MicroRNAs 218a-5p, 219a-5p, and 221-3p regulate vestibular compensation.</a> Scientific Reports. 7 (1), 8701 (2017).</li><li>Bergquist, F., Ludwig, M., Dutia, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+commissural+inhibitory+system+in+vestibular+compensation+in+the+rat.">Role of the commissural inhibitory system in vestibular compensation in the rat.</a> The Journal of Physiology. 586 (18), 4441-4452 (2008).</li><li>Cameron, S. A., Dutia, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+vestibular+compensation:+changes+in+intrinsic+excitability+of+MVN+neurones.">Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones.</a> NeuroReport. 8 (11), 2595-2599 (1997).</li><li>Park, M. K., Lee, B. D., Lee, J. D., Jung, H. H., Chae, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+profiles+during+vestibular+compensation+in+rats+after+unilateral+labyrinthectomy.">Gene profiles during vestibular compensation in rats after unilateral labyrinthectomy.</a> Annals of Otology, Rhinology &amp; Laryngology. 121 (11), 761-769 (2012).</li><li>Yamamoto, H., Tominaga, M., Sone, M., Nakashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+stapedial+artery+to+blood+flow+in+the+cochlea+and+its+surrounding+bone.">Contribution of stapedial artery to blood flow in the cochlea and its surrounding bone.</a> Hearing Research. 186 (1-2), 69-74 (2003).</li><li>Potegal, M., Abraham, L., Gilman, S., Copack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+vestibular+neurotomy+in+the+rat.">Technique for vestibular neurotomy in the rat.</a> Physiology &amp; Behavior. 14 (2), 217-221 (1975).</li><li>Tung, V. W., Burton, T. J., Dababneh, E., Quail, S. L., Camp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+assessment+of+the+aging+mouse+vestibular+system.">Behavioral assessment of the aging mouse vestibular system.</a> Journal of Visualized Experiments. (89), e51605 (2014).</li><li>de Jeu, M., De Zeeuw, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-oculography+in+mice.">Video-oculography in mice.</a> Journal of Visualized Experiments. 65 (65), e3971 (2012).</li></ol>

著者が明らかに何もありません。

この手法は、突然、恒久的なかつ完全な前庭機能の損失を作成する便利な方法です。これは、前庭神経炎、聴神経腫瘍やメニエール病などの前庭疾患を勉強される可能性があります。多くの研究は、前庭神経核の神経細胞の可塑性や関連する中央プロセス5,17,18,19を研究するのにこの手法を使用し...

前庭器官は、バランスと目のコントロールは欠かせません。通常の前庭機能は、2 つの内側の耳の前庭器官から対称の求心性信号によって異なります。前庭機能低下または損失は、めまい、眼振、姿勢の不均衡を誘導します。急性損傷後前庭機能が自発的に回復数日前庭代償1,2として知られているプロセス。静的な赤字の前庭代償は、安静時自発同側および対側の前庭神経核間の不均衡に関連する回復のプロセスです。動的赤字の前庭代償を達成した主を介して感覚と行動置換 (視覚や体性感覚の入力を使用して)3。これらのプロセスが神経可塑性研究4、5の魅力的です。
前庭系と前庭代償、外科および化学なるほどなど前庭神経切断5,6 中神経可塑性を支えるメカニズムを検討するいくつかの方法が開発されています。 ,7,8。前庭神経切断完了の前庭系障害を誘発する特定の方法が難しく、侵襲的なプロシージャで、脳損傷8,9を引き起こす可能性があります。このメソッドは、高度な外科的スキルが必要です、なるほどより時間かかります。ゲンタマイシン、arsanilate、テトラカインなど化学なるほどが簡単で信頼性の高い結果10、11,12をもたらすことができます。しかし、蝸牛が壊れているも可能性が、前庭系障害時間11に発展するかもしれない。さらに、化学物質の正確な評価のために維持されるべき、脳に及ぼす影響は明確ではありません。手術なるほど 184215の動物研究で初めて導入されました、1936年16でラットに初めて報告されました。この手法が多く使われており動物研究5,17,18,19。手術なるほど、特定の信頼性の高い、比較的単純な方法であります。13,14また、前庭障害の症状は、手術後すぐに見られています。ここでは、ラットなるほどに対する私たちの手技について述べる。

<table><tbody><tr><td>ASPIRATOR KB-012</td><td>KOH BONG &amp;amp; CO., LTD.</td><td>KB-012</td><td>Medical aspirator</td></tr><tr><td>Blade: #15</td><td>Fine Science Tools</td><td>#10015-00</td><td>Blades for #7 Scalpel Handles, #15</td></tr><tr><td>Carbon Steel Burrs</td><td>Fine Science Tools</td><td>#19007-05</td><td>shaft diameter: 2.3 mm, length: 44 mm, package of 10 burrs</td></tr><tr><td>Carl Zeiss Surgical GmbH</td><td>Carl Zeiss</td><td>#6627100863</td><td>Surgical microscope</td></tr><tr><td>Dumont #3c</td><td>Fine Science Tools</td><td>#11231-20</td><td>Standard tip 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Dumont #5SF</td><td>Fine Science Tools</td><td>#11252-00</td><td></td></tr><tr><td>Dumont #7B</td><td>Fine Science Tools</td><td>#11270-20</td><td>Serrated 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Extra Fine Bonn: straight</td><td>Fine Science Tools</td><td>#14084-08</td><td>Iris scissors, best suited for microdissection under high magnification</td></tr><tr><td>Fine Iris Scissors: straight</td><td>Fine Science Tools</td><td>#14094-11</td><td>Made from martensitic stainless steel, combined with molybdenum and vanadium</td></tr><tr><td>Finger Loop Ear Punch</td><td>Fine Science Tools</td><td>#24212-01</td><td>1 mm. Provides stability and control for researchers using the numbering system</td></tr><tr><td>Hartman</td><td>Fine Science Tools</td><td>#13002-10</td><td>Tip width: 1 cm, serrated, 10 cm</td></tr><tr><td>Short Scalpel Handle #7 Solid</td><td>Fine Science Tools</td><td>#10003-12</td><td>#7 short, 12 cm</td></tr><tr><td>Small Vessel Cauterizer</td><td>Fine Science Tools</td><td>#18000-03</td><td>Replacement tip, straight knife, keeps bleeding to a minimum and therefore provides a surgical field clear of clamps and hemostats</td></tr><tr><td>Strong 207S</td><td>SAESHIN</td><td>207S</td><td>Powerful torque at low speed, available with speed or on/off foot controller</td></tr><tr><td>Suction Tubes</td><td>JEUNGDO B&amp;amp;P CO., LTD.</td><td>H-1927-8</td><td>Frazier, 18 cm</td></tr><tr><td>VICRYL</td><td>ETHICON</td><td>W9570T</td><td>Synthetic absorbable sterile surgical suture</td></tr><tr><td>Weitlaner-Locktite</td><td>Fine Science Tools</td><td>#17012-13</td><td>Maximum spread: 4.5 cm, 2 x 3 blunt teeth, 11 cm</td></tr><tr><td>Zoletil</td><td>Virbac, France</td><td></td><td>Tiletamine-zolazepam</td></tr><tr><td>Rompun</td><td>Bayer</td><td></td><td>Xylazine</td></tr><tr><td>Rimadyl</td><td>Pfizer</td><td></td><td>Carprofen</td></tr><tr><td>Septra</td><td>Pfizer</td><td></td><td>Trimethoprim-sulfonamide&amp;nbsp;</td></tr></tbody></table>

surgical labyrinthectomy of the rat to study the vestibular system

前庭系や前庭代償プロセスを研究するため、メソッド数は外科か化学なるほどと前庭神経切断などを含む前庭神経の損傷を引き起こすことしました。手術なるほどは、比較的単純な信頼性、および迅速な方法です。ここでは、ラットなるほどのために手術手技について述べる。耳介後部切開が外耳道と鼓膜、鼓膜と小骨があぶみ骨なし削除される公開全身麻酔下で行われます。あぶみ骨と楕円形の窓との間に位置しているアブミ骨動脈は脆弱な構造を明確な外科的フィールドを取得する保持必要があります。かぎり行うホール前庭で行われます 2.1 mm のドリル バールあぶみ骨に優れた。100% エタノールはこの穴から注入し、数回を吸気します。綿密な解剖顕微鏡および注意深い出血制御の下で、信頼性の高い結果を得るために不可欠です。手術直後後は、前庭系障害、眼振、頭の角度、回転運動などの症状が見られます。Rotarod または回転椅子テストは、客観的・定量的評価前庭機能を使用できます。

手術の成功は、行動テストによって検証されました。すべての動物は、片側性前庭機能低下の典型的な動作を示した。自発的なバレル ローリングは、早期復旧フェーズ (ビデオ 1) で頭や体を軽いタッチで空気のパフで誘発されて、手術後すぐに明白でした。手術後 3 d は、時折破壊側に傾いて移動動物は左側に落ちる。自発眼振は、2 d (動画 2</strong...

Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

このプロトコルでは、前庭系を研究するための有用な方法であるラットの手術なるほどについて説明します。

前庭系を研究するラットの手術なるほど

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

surgical-labyrinthectomy-of-the-rat-to-study-the-vestibular-system

Research

JoVE Journal

Medicine

10.0K ビュー.  Chung-Ang University College of Medicine. このプロトコルでは、前庭系を研究するための有用な方法であるラットの手術なるほどについて説明します。

ビデオ: Surgical Labyrinthectomy of the Rat to Study the Vestibular System

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

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

げっ歯類で存続可能な定位手術

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

プリザーブドラットの采 - 脳弓の病変後に香りのトラッキングが障害推測航法による操縦のビデオデモ

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

エレベーター垂直運動と観覧車回転を用いたラットにおける受動運動の自律性と行動の影響の評価

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

行動学

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

電気生理学および高分解能二光子顕微鏡用マウス前庭感覚上皮の切り抜き半無傷準備

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

神経科学

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由に行動するマウスで長期的な感覚の衝突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

テトロドトキシンマイクロインジェクションによる可逆不活性化による排卵調節におけるラット脳の離散領域の役割の解明

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

近赤外（NIR）の光は、マウス前庭感覚上皮におけるミトコンドリア機能のマーカーの発現を増加させる

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

パッチクランプ記録のための新生児げっ歯類からの前庭およびらせん状神経節体細胞の分離と培養

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.