私たちは、優雅に、データ収集（アンドリース·フェレイラ、Danaraeアレマン、アレクシスモク、Kaitlinフリン、エリザベスBearce、およびマタンKadosh）と原稿レビュー（アンドリース·フェレイラ、レベッカシュナイダー、そしてケイト·ロビンズ）に貢献したレバーラボの追加のメンバーに感謝。また、本研究で用いたげっ歯類の観測管の彼らの設計入力および製造のためのMU物理マシンショップからロ​​デリックSchlotzhauerとエドウィンHonseを認める。とヤンアイビー（ミズーリ大学の研究動物キャスラボのマネージャー - 医学部） - 私たちは、Maleaヤンクンケル（獣医学の大学ミズーリ大学の獣医医療および外科手術部門の放射線スーパーバイザー）の特に感謝してい我々はネズミVFSSプロトコルを開発したとして、高エネルギー透視を動作させながら一定の忍耐と意欲を実証するため。この研究のための資金源は、NIH / NIDCD（TEレバー）、NIH / NINDS（GK Pavlath）、Otolaryngoを含まLOGY - 頭頸部外科起動資金（TEレバー）、MU PRIME基金（TEレバー）、ミゾーアドバンテージ（TEレバー）、及びエイジング上のMUセンター（TEレバー）。

この記事のオープンアクセスはGlenbrookのが主催している。

（減損嚥下）嚥下障害は、すべての年齢層の人々に影響を与え、多くの病状の一般的な症状です。例としては、脳卒中、パーキンソン病、アルツハイマー病、脳性麻痺、筋ジストロフィー、筋萎縮性側索硬化症（ALS）、バッテン病、頭頸部癌、早産、および高度な老化を含む。嚥下障害は非常に一般的に細菌を含んだ食品/液体/唾液が肺に1-4内に吸引されたときに発症する重度の栄養失調や肺炎の結果として、死亡率と相関している。この衰弱と生命を脅かす医学的状態は、米国だけで3には毎年1500万人に影響を与えます。有病率が高いとそれに関連する否定的な結果にもかかわらず、嚥下障害のための現在の治療法の選択肢は、（むしろ治癒なく）姑息に限定されている（ 例えば、特定の食品/液体コンシステンシーを回避する）ようなダイエット変形例として、近づくと、姿勢の変化（ 例えば、tucki）、モータのアプローチ（ 例えば、エクササイズ）、口腔、咽頭、および喉頭の筋肉を標的とする、感覚的なアプローチ（ 例えば、実施味、温度、および/ ​​または機械的刺激）を飲み込むときに顎ngを、そして管栄養（ 例えば、栄養と水和は経鼻胃（NG）チューブまたは経皮的内視鏡的胃瘻造設術（PEG）チューブ）を介して投与。これらの治療は、単にむしろ、問題の根本的な原因をターゲットによりなど対症療法を提供する。実際、小説、嚥下障害のための効果的な治療法の発見に大きな障壁は、各疾患のための可能性が異なっている責任の病理学的メカニズム、の限られた科学的な知識である。 嚥下障害の診断は、主にも修正されたバリウム嚥下研究として知られるvideofluoroscopic嚥下研究（VFSS）、と呼ばれるX線撮影の手順を使用して行われる。過去30年以上にわたり、この診断テストは、EV用のゴールドスタンダードを検討されているツバメ機能5-7 aluating。このテストは、典型的には、硫酸バリウム8,9またはイオヘキソール10は、患者が座ったり自発的に食品や経口造影剤と混合された液体の稠度を摂取しながら、X線透視機のX線ビームの経路内に立ったことを伴う。患者が飲み込むように口から胃へ移動しながら、食品や造影剤を含有する液体は、コンピュータモニタを介してリアルタイムで見ることができる。軟組織構造も表示され、構造および機能に関連して評価することができる。患者は、嚥下障害の有無と程度を定量化するために、その後の表示やフレームごとの解析のために記録された映像であるそのすべてが、それぞれの食品や液体の一貫性のいくつかのツバメを実行するように求められます。嚥下の多くの生理学的成分は、典型的には、larynの咽頭や食道、程度及び持続期間を通じて、そのような咽頭ツバメの解剖トリガーポイントとして、ボーラス通過時間を分析し、geal標高、場所およびポストツバメの残留物の量、およびの発生及び吸引7,11のための生理的な理由。 人間VFSSプロトコルの態様は、最近になって、自由に振る舞うラットを研究するために適応した。ラットは12のテスト中に、ビューのvideofluoroscopic視野に残っていなかったので、結果が制限されていました。 VFSSは以前にマウスと試みられていない。マウスおよびラットでの使用のためのヒトVFSSプロトコルの成功した適合は、現在、既存のマウス（マウスおよびラット）、ヒトにおける嚥下障害を引き起こすことが知られている疾患のモデルの数百を調べるための新規な研究方法を提供する。この新しい方法（以下マウスVFSSとも呼ばれる）は、従って病的である、筋肉、神経、脳組織内の基礎となる神経生理学的メカニズムを調査し、嚥下障害に寄与するのに適している嚥下障害のマウスモデルの同定および確認を早めるだろう私nの人間。また、マウスVFSS直接人間と比較することができる嚥下機能/機能不全の客観的尺度（バイオマーカー）の同定を可能にする。これらの異種間videofluoroscopicバイオマーカーは、その後、より良い人々との臨床試験に翻訳するでしょう、マウスやラットを用いた前臨床試験で治療効果を定量化するための新規なアウトカム指標を、役立つことができる。 このために、ネズミVFSSプロトコルはセックスのどちらかの〜100マウスを使用して設立されました。すべてのマウスは、C57またはハイブリッドC57 / SJL株のいずれかであった。 C57 / SJLトランスジェニックSOD1-G93A（またはSOD1）のコロニーのバックグラウンド株であったのに対し、C57マウスは、ALSの最も広く使用されている動物モデルマウスの遺伝的に改変されなかった。 SOD1のコロニーは、トランスジェニック（ すなわち、ALSの影響を受けた）マウスおよび非トランスジェニック（ すなわち、影響を受けない）同腹仔のおおよそ50-50ミックスだった。 ネズミVFSSプロトコルは、3つのコンポーネントで構成されています。 <ol><lI&gt; X線透視機内に閉じ込め空間に拘束されていない立っている間自発的摂食と嚥下を許可するカスタム設計された観測室、 </li><li>経口造影剤の嫌悪味/臭いをマスクし、嚥下の適切な可視化を可能にするのに十分な放射線濃度を生成するレシピ</li><li>動物のコンプライアンスを最大化するステップバイステップの試験プロトコルは、総試験時間および放射線被曝を最小限に抑え、かつ（ すなわち、口腔、咽頭、食道）の嚥下の各段階のためのいくつかの嚥下パラメータの定量化を可能にする。 </li></ol>複合効果は、典型的な給餌マウスの嚥下行動の評価を可能にする快適な、低応力、自己給電検査環境を生成する。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >Polycarbonate tubing for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >3161T41</td>
			<td>Body of observation tubes, 2&#34;X2&#34; diameter, 0.080&#34; thick wall</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9115K71</td>
			<td>End-caps for observation tubes, 2&#34;x12&#34;x3/4&#34;</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >8574K281</td>
			<td>Peg-bowls for observation tubes</td>
		</tr>
		<tr >
			<td >Silicone O-rings&#160; for end-caps of observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9396K108</td>
			<td>S1138 AS568-029, pack of 25 
			http://www.mcmaster.com/#o-rings/=t0wt5r&#160;</td>
		</tr>
		<tr >
			<td >Silicone stoppers for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >2903K22</td>
			<td>Package of 10 stoppers to plug the oval opening in the top of the observation chamber when using a peg-bowl 
			http://www.mcmaster.com/#catalog/120/3803/=t0y5at</td>
		</tr>
		<tr >
			<td >Centrifuge tubes for sipper tube bottles</td>
			<td >Evergreen Scientific</td>
			<td >222-3530-G80</td>
			<td>30 ml freestanding centrifuge tubes, with caps, sterile 
			https://www.evergreensci.com/labware-catalog/tubes-and-vials/30-and-50-ml-centrifuge-tubes/&#160;</td>
		</tr>
		<tr >
			<td >Silcone stoppers for sipper tube bottles</td>
			<td >Saint-Gobain Performance Plastics</td>
			<td >DX263031-10&#160;</td>
			<td>Number 31D, size: 26 mm bottom, 32 mm top, 30 mm high; 10 pack;&#160; 
			http://www.labpure.com/en/Products.asp?ID=179&#38;PageBrand=STOPPERS</td>
		</tr>
		<tr >
			<td >Stopper borers for sipper tube bottles</td>
			<td >Thomas Scientific</td>
			<td >3276G40</td>
			<td>Cork Borer Set that ranges from 3/16-15/16 inch&#160; 
			http://www.thomassci.com/Supplies/Corks/_/CORK-BORER-SET-316-1516-IN?q=Humboldt</td>
		</tr>
		<tr >
			<td >Drinking tubes for sipper tube bottles</td>
			<td >Ancare</td>
			<td >TD-100&#160;</td>
			<td>2 1/2&#8221; long drinking tubes with 5/16&#8221; opening, straight ball-spout 
			http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point&#160;</td>
		</tr>
		<tr >
			<td >Iohexol for making oral contrast agent solution</td>
			<td >GE Healthcare</td>
			<td ></td>
			<td>350 mg iodine per ml 
			http://www3.gehealthcare.com/en/products/categories/contrast_media/omnipaque&#160;</td>
		</tr>
		<tr >
			<td >Chocolate syrup for flavoring oral contrast agent</td>
			<td >Herseys</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >10 ml syringe for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >309604</td>
			<td>Luer lock tip syringe without needle, 100 per box 
			http://www.bd.com/hypodermic/products/syringeswithoutneedles.asp</td>
		</tr>
		<tr >
			<td >Catheter tubing for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427451</td>
			<td>Polyethylene Tubing (Non-Sterile) (PE 240) 100&#39; 
			http://www.bd.com/ds/productCenter/427451.asp&#160;</td>
		</tr>
		<tr >
			<td >Needle for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427560</td>
			<td>15-gauge needle, fits into PE 240 catheter tubing 
			http://www.bd.com/ds/productCenter/427560.asp&#160;</td>
		</tr>
		<tr >
			<td >Delrin acetal resin rod for syringe delivery system</td>
			<td >McMaster-Carr</td>
			<td >8576K15</td>
			<td>1/2 inch diameter, black 
			http://www.mcmaster.com/#catalog/120/3609/=t0wvaf&#160;</td>
		</tr>
		<tr >
			<td >Acrylic sheeting for scissor lift</td>
			<td >Ponoko</td>
			<td ></td>
			<td>Laser cut 
			http://www.ponoko.com&#160;</td>
		</tr>
		<tr >
			<td >3D printed ABS frame</td>
			<td >Engineering Rapid Prototyping Facility, University of Missouri</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Brass rods for scissor lift</td>
			<td >Amazon</td>
			<td >TTRB-03-12-03</td>
			<td>made into axles 
			http://www.amazon.com/Brass-Seamless-Round-Tubing-Length/dp/B000FN898M</td>
		</tr>
		<tr >
			<td >Drawer slide for scissor lift</td>
			<td >Richelieu</td>
			<td >10292G116</td>
			<td>Attaches to base of scissor lift 
			http://www.lowes.com/pd_380986-93052-T35072G16_0__?productId=50041754</td>
		</tr>
		<tr >
			<td >28BYJ-48 stepper motor for scissor lift</td>
			<td ></td>
			<td ></td>
			<td>2 each</td>
		</tr>
		<tr >
			<td >ULN2003 Darlington transistor array for scissor lift</td>
			<td >Toshiba</td>
			<td >ULN2003APG</td>
			<td>Used as stepper drivers (2 each)</td>
		</tr>
		<tr >
			<td >ATTINY85 microcontroller for scissor lift</td>
			<td >Atmel</td>
			<td >ATTINY85-20PU</td>
			<td>2 each 
			http://www.taydaelectronics.com/attiny85-attiny85-20pu-8-bit-20mhz-microcontroller-ic.html</td>
		</tr>
		<tr >
			<td >Nylon spur gear</td>
			<td >McMaster-Carr</td>
			<td >57655K34</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k34/=t0yaqz</td>
		</tr>
		<tr >
			<td >Nylon spur gear rack</td>
			<td >McMaster-Carr</td>
			<td >57655K62</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k62/=t0ybh9</td>
		</tr>
		<tr >
			<td >4-40 nylon machine screws</td>
			<td >McMaster-Carr</td>
			<td >95133A315</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#95133a315/=t0yd8q</td>
		</tr>
		<tr >
			<td >4-40 nylon hex nuts</td>
			<td >McMaster-Carr</td>
			<td >94812A200</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#94812a200/=t0ye29</td>
		</tr>
		<tr >
			<td >Buna-N O-Ring AS568A Dash No. 104</td>
			<td >McMaster-Carr</td>
			<td >9452K318</td>
			<td>Lift assembly 
			http://www.mcmaster.com/#9452k318/=t0yem7</td>
		</tr>
		<tr >
			<td ></td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

adapting human videofluoroscopic swallow study methods to detect and characterize dysphagia in murine disease models

この研究は、トランスレーショナ嚥下障害の研究を促進する目的で、マウス疾患モデルで使用するための研究（VFSS）メソッドを飲み込む人間videofluoroscopicに適応。その限られたスペースで気ままに立っている間に自己供給を許可した試験室、市販の経口造影剤の嫌悪味/臭いをマスクするレシピ、ステップバイステップの試験プロトコル：成功の結果は3つの重要なコンポーネントに依存している嚥下生理学の定量を可能にする。これらのコンポーネントのうちの1つまたは複数の除去は、試験結果に悪影響を与えるであろう。また、X線透視システムのエネルギーレベル能力を調べることができる飲み込むパラメータ決定する。ほとんどの研究センターでは、マウスや他の小型げっ歯類をテストする際に非常に悪い画質になり、人とより大きな動物での使用のために設計された高エネルギー透視を持っている。この制限にもかかわらず、私たちは7 VFSを特定した新しいマウスVFSSプロトコルと組み合わせて、高エネルギーX線透視装置を使用した場合、マウスにおいて一貫して定量可能であるSパラメータ。我々は最近、マウスなどの小型げっ歯類で使用するために設計された非常に高い結像分解能と倍率機能と低エネルギーX線透視システムを得た。この新しいシステムを使用する準備作業は、新しいマウスVFSSプロトコルと組み合わせて、従来の（すなわち、高エネルギー）透視装置を用いて得られ、ほぼ倍の数であるマウスにおいて一貫して定量可能である13ツバメパラメータを特定した。我々は、この新しいシステムの機能を最適化するように追加の嚥下パラメータの同定が期待されている。結果は、これまでのマウス疾患モデルを調査するために、高エネルギー透視装置を使用する際に見過ごされてもよい嚥下生理学における微妙な変化を検出し、定量化するために、低エネルギーX線透視システムを使用しての有用性を実証する。

我々は、試験の自己給電を可能にするチャンバ、経口造影剤、香味剤のレシピ、および嚥下生理学の定量を可能にするステップバイステップの試験プロトコルを含む新規かつ複製可能なマウス固有VFSSプロトコルを成功裏に設計した。透視システムのエネルギーレベル機能は、パラメータがマウスにおいて調査することができる飲み込むが決定。私たちは、最初は人で使用するために設計された高エネルギー透視、より大きな動物（ 例えば、GE Advantx、GE OEC 9600、およびオメガ心臓カテーテル検査CS-25、毎秒30フレームの各）を使用した。しかしながら、これらのシステムは、動物の充填で、視野のわずかな部分をもたらした、試験マウスには不十分な拡大機能を有していた （ 図9）。その結果、画質は不可能嚥下機構のほとんどの構造を視覚化する、レンダリング、非常に不良であった。この制限にもかかわらず、私たちは7客観VFSを特定しSは、新しいマウスVFSSプロトコル（ 表3）と組み合わせて、従来の（ すなわち、高エネルギー）蛍光透視鏡を使用した場合のマウスについて一貫して定量可能であったパラメータを飲み込む。さらに、我々は高齢（&gt; 18ヶ月）と末期ALSの条件vallecularの健康な成体マウスに嚥下解剖トリガ·ポイントとして空間（生後3-17ヶ月）、ならびにマウスを同定した。 <img alt="図9" src="/files/ftp_upload/52319/52319fig9highres.jpg" width="650" /> 図9：。高エネルギーX線透視システム左：高エネルギー（ すなわち、従来の）X線透視システムを使用して得られたマウスの代表的なイメージ。マウスは、それによってイメージングげっ歯類のための従来の透視の不十分な倍率能力を実証する、ビューのX線透視視野のごく一部を埋めることに注意してください。右：同じ画像拡大ポストcaptuビデオ編集ソフトウェアプログラムを使用して再。黒矢印：トリガーポイント（valleculae）を飲み込む。白矢印：遠位食道内ボーラス、GE接合（白アスタリスク）を通過する直前に<a href="https://www.jove.com/files/ftp_upload/52319/52319fig9highres.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> SWALLOWパラメータ</td><td style="width:140px">高エネルギーシステム</td><td style="width:140px">低エネルギーシステム</td></tr><tr><td>インターツバメ間隔（ISI） </td><td> X </td><td> X </td></tr><tr><td>ジョーエクスカーションレート（リック·レート換算） </td><td> X </td><td> X </td></tr><tr><td>ジョーエクスカーション距離</td><td> X </td><td> X </td></tr><tr><td>リック·ツバメ比</td><td> X </td><td> X </td></tr><tr><td>ツバメレート</td><td> X </td><td> X </td></tr><tr><td> PharyngEALトランジット時間（PTT） </td><td></td><td> X </td></tr><tr><td>咽頭を通してボーラススピード</td><td></td><td> X </td></tr><tr><td>食道トランジット時間（ETT） </td><td></td><td> X </td></tr><tr><td>食道を通っボーラススピード</td><td></td><td> X </td></tr><tr><td>咽頭と食道を通っボーラススピード</td><td> X </td><td> X </td></tr><tr><td>ボーラスエリア</td><td></td><td> X </td></tr><tr><td>咽頭残留エリア</td><td></td><td> X </td></tr><tr><td>液体の体積が消費</td><td> X </td><td> X </td></tr></tbody></table> 表3：低エネルギーX線透視システムバーサスハイを使用してパラメータ定量化スワロー。 我々は最近、特にで使用するために私たちの研究室のために設計されましたLabScope（Glenbrookのテクノロジーズ、ランドルフ、ニュージャージー州）と呼ばれる低エネルギー倍率X線透視システムを得マウスなどの小型げっ歯類（ 図10）。しかし、このシステムの顕著に大きな倍率レベルは、それが不可能な単一の視野でのマウスの全体嚥下メカニズムを表示するレンダリング。 図11の位置1を許可するヘッド全体の可視化および近位胸部に示されるように代わりに二つの試験位置は、必要とされる。この位置は、嚥下の口腔咽頭の段階を評価するために必要である。胃食道（GE）接合部にスワロートリガ点（ すなわち、valleculae）からポジション2許可の可視化。この位置は、嚥下の食道段階を評価するために必要である。新しいマウスVFSSプロトコルと組み合わせLabScopeを用いた予備研究は、高エネルギー（ すなわち、従来の）透視した（ 表3）を用いて得倍近い数である、マウスにおいて一貫して定量化され13、対物嚥下パラメータを特定した。このO<html> 例えば、舌骨、気管、および頸椎：utcomeは、従来のシステムを使用する場合、本質的に見えなかった多数の解剖学的構造の可視化を可能にするLabScope、（ 図12）の高度な拡大能力に起因する。その結果、我々はまた、喉頭侵入や誤嚥の証拠ビデオを分析することができました。浸透も誤嚥どちらもかかわらず、健康や病気の状態の、この研究では任意のマウスを観察した。 <img alt="図10" src="/files/ftp_upload/52319/52319fig10highres.jpg" width="650" /> 図10：。LabScope左：LabScopeは小動物用のデスクトップ透視装置として実行されます。右：標識成分とのLabScopeのクローズアップ図。シザーリフトテーブル、ビューのX線透視フィールド内の観察室を位置づけている。<html> tp_upload / 52319 / 52319fig10highres.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図11" src="/files/ftp_upload/52319/52319fig11highres.jpg" width="650" /> 図11：低エネルギーX線透視システムを使用して得られたマウスの低エネルギーX線透視システムイメージ。高倍率能力がビューのX線透視視野内全体のツバメ機構の可視化を妨げることに注意してください。左：ポジション1は - 全体の頭と近位胸部領域の可視化を可能にする。嚥下トリガー点（黒矢印）は、本質的に視野内の中心にある。右：ポジション2 - スワロートリガーポイント（黒矢印）からGE接合（白アスタリ​​スク）に可視化を可能にします。遠位食道（白矢印）を通過ボーラスに注意してください。<html> g11highres.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図12" src="/files/ftp_upload/52319/52319fig12highres.jpg" width="650" /> 図12：低エネルギー透視システムを使用して、解剖学的構造の可視でも最低の倍率設定では、（左）、マウスの頭と首のボニー構造は、私たちの低エネルギーX線透視システム（ すなわち、LabScope）を用いて容易に見ることができます。黒い四角内の解剖学的構造を示す（およびラベル）を右に、より高い倍率でされている。ボニー構造の改善された可視化は、高エネルギー透視を使用して分析することが不可能であったいくつかの追加ツバメパラメータの定量化を可能にします。 <a href="https://www.jove.com/files/ftp_upload/52319/52319fig12highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> tは&quot;&gt;率とインター嚥下間隔が新しいマウスVFSSプロトコルと組み合わせて低または高エネルギーX線透視システムのいずれかを使用して定量化することができる代表的なVFSSパラメータであるスワローこれら二つの嚥下パラメータは3群のマウスについて定量した：SOD1-G93A疾患における（すなわち、ALSのモデル）（SOD1）トランスジェニックマウスは、年齢、老化C57マウス（生後18-24ヵ月）の4-5ヶ月の間の末期、及び健康な若い（4-8ヶ月の対照群C57マウスとSOD1コロニーから非トランスジェニック同腹仔）年齢。すべてのデータは、低または高エネルギーX線透視システムのいずれかを使用して、唯一の飲酒を噴出するために関係する。有意差はSOD1から若いC57マウスと若い非トランスジェニック（コントロール）マウスの間で見られなかったすなわち 、したがって、データは高齢のC57マウスと末期SOD1マウスとの比較のために、若い健康なマウスの一般的な「制御」のグループに合わせた率スワロー（;。これら二つのツバメパラメータへのコロニーの相対途切れない飲酒の2秒間連続して）中のツバメの数は、高齢のC57マウスと対照と比較して、SOD1マウスで有意に遅かった。インターツバメ間隔（すなわち、2つの連続したツバメの間の時間）は、群間で有意差はなかった。これらの知見は、嚥下障害のプロファイルは、各疾患状態（ 図13）のための明確に異なる可能性があるという考えを支持する。 <img alt="図13" src="/files/ftp_upload/52319/52319fig13highres.jpg" width="650" /> 図13：予備調査結果は、この図は、ネズミVFSSプロトコルを使用して定量2 VFSSスワローパラメータの代表予備調査結果を示しています。レート（左）とインターツバメ間隔（右）飲み込む。スワロー率は高齢のC57マウスと対照と比較して、SOD1マウスで有意に遅かった。有意な群差は、インターツバメの間のために確認されなかったヴァル。バーの上部の行は、ボンフェローニのペアワイズ比較を用いて同定されたグループの間に統計的に有意な差（p &lt;0.05）を示す。エラーバーは±1 SEMを表す。 <a href="https://www.jove.com/files/ftp_upload/52319/52319fig13highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models at JoVE.com

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

この研究は、成功したトランスレーショナ嚥下障害の研究を促進する目的で、マウス疾患モデルで使用するための人間のvideofluoroscopic嚥下研究（VFSS）メソッドを適応さ。

マウス疾患モデルで嚥下障害を検出し、特徴づけるために、人間のVideofluoroscopicツバメ調査方法を適応させる

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

adapting-human-videofluoroscopic-swallow-study-methods-to-detect

Research

JoVE Journal

Medicine

21.0K ビュー.  University of Missouri. この研究は、成功したトランスレーショナ嚥下障害の研究を促進する目的で、マウス疾患モデルで使用するための人間のvideofluoroscopic嚥下研究（VFSS）メソッドを適応さ。 

ビデオ: Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

人間の不完全なSCIにおける運動機能の薬理学的に誘起される変化を定量化する方法

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

医学

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

正常なヒト脳内の可溶性および不溶性のPrPオリゴマーの分離

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

多発性嚢胞腎疾患で、ヒトとマウスの染色体異常を研究するためにスペクトル核型分析

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

卵巣がん再発を検出し、モニターするための非侵襲的イメージングのためのマウスモデル

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

単離とマウス大動脈の切除。心血管疾患の研究で多彩なテクニック

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

急性および亜急性ネズミ後肢虚血のための方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

塩化第二鉄誘発性マウス血栓症モデル

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...