This work is funded by NIH RO1 NS 40433 (K.J.J.).

実行されるすべての手順は、医療機関動物実験委員会のインディアナ大学大学院によって承認され、健康ガイドラインの国立研究所に従っている。 1.手術手技<ol><li> NIHガイドライン25に従って無菌滅菌手袋を使用して、この手順の間に技術、機器、および無菌手術野を維持します。 （完全なリストに特異的な試薬/機器の表を参照）、それらをオートクレーブで手術を開始する前に、ツールを滅菌する。動作中のツールを殺菌するためにガラスビーズ滅菌器を使用してください。 </li></ol> 2.麻酔と準備<ol><li>獣医イソフルラン気化器システムを用いて、0.9リットル/分の酸素と2.5％のイソフルランの混合物で麻酔ボックス内にマウスを麻酔。マウスを箱から取り出す前に、身体の位置の変化に応答しないことを確認してください。 </li><li> MOUに眼軟膏を適用します乾燥からそれらを保護するために、それ自体の目。 </li><li>ノーズコーンにあるボックスからのガスの流れを切り替えます。コー​​ンの内側の鼻と口を備えた外科用パッドと吸収ベンチ紙で覆われて加熱されたパッドの上に真正面からその左側にマウスを置きます。継続的にマウスの呼吸のリズムと速度を監視し、必要に応じてイソフルランレベルを調整 - 麻酔の適切なレベルを維持するために、（2.5の間で3％イソフルラン）、および総鎮静を確認するために、つま先ピンチ反射を使用しています。 </li></ol> 3.外科的アプローチ<ol><li>合わせて、手術野でステレオスコープを集中。それは視野の端に沿って配置されるように、ノーズコーンとテープを上下に調整する。 </li><li>マウスは、その左側に横たわっていると、切開が行われる耳の後ろの領域を露出させ、ノーズコーンに右耳の端をテープで固定します。後耳介静脈が耳全体に水平に移動することを確認してください。 Tの正しい配置に注意してください彼は耳の動物とテーピングはすぐに顔面神経を見つけるために重要である。 </li><li>にし、70％エタノールで耳の後ろに毛皮を湿らせ、かみそりまたは外科用メスの刃を使用して、手術部位を剃る。毛皮を事前に湿潤この解剖学的位置に簡単に剃ることができます。 </li><li> 70％エタノールに続くようなベタジン外科スクラブ（7.5％ポビドンヨード）などのヨウ素溶液、で皮膚をきれいにしてください。徹底的に領域を消毒するために、この清掃をさらに2回繰り返します。 </li><li> 、切開する耳の突起にエリア後方に尾側耳から後耳介静脈をトレースする場所を決定する。隆起に3ミリメートルの後部 - 春のはさみを使用して、4ミリメートル切開2を作る。 </li><li>鈍的切開を使用して皮下脂肪および筋膜を通して解剖。 、血管または筋肉組織を簡単に破損する可能性があるため、ハサミで直接切断しないでください。 </li><li>出血が発生した場合は、無菌の綿棒で手術部位に圧力を適用する少なくとも30秒間。重要な流体損失が発生した場合、25または27 G針を使用して0.9％滅菌生理食塩水までの0.5mlを腹腔​​内にマウスを注入する。 </li><li>顔面神経を見つけるために、いくつかの主要なランドマーク、脊髄副神経、外耳道を使用し、（後述）二腹筋を前方。顔面神経の枝が可視化されるまで、これらのランドマークの周りに解剖。それは明らかにし、筋膜の層が下にある構造に付着されたときに神経が重要な白色固体構造として表示されます。 <ol><li>皮下脂肪と筋膜が解剖された後、僧帽筋を支配するために頭蓋骨の尾の部分から移動する脊髄副神経を検索します。顔面神経は、脊髄アクセサリ神経に深い。 </li><li>真珠のような白に見え、顔面神経への吻側見ることができる軟骨外耳道を探す。 </li><li>の上部とC上に位置し、前二腹筋の筋腹を探す顔面神経にaudal。 </li></ol></li><li>顔面神経の主枝を可視化されると、茎乳突孔からその起源を見つけるために背側にそれらをトレースします。細かい使用すると、開いて新しく高度な面積を維持するために背側鉗子を移動した後、開いた手術部位を保持する神経のパス以下の春のはさみのヒントを進めるためにデュモン鉗子＃45分の5をひっくり返した。 </li><li>この時点で、頬骨と顔面神経の幹、頬、および限界下顎枝を可視化する。 注：一時的な分岐が孔に近い見つかります。近い顎にその上下に限界下顎神経の枝、従って、これらの神経枝は、このレベルでは表示されません。 <ol><li>神経切断を行う場合は、先端の細い鉗子で軽く神経を安定させ、春のはさみで神経を切った。脳幹から神経をavulsing防ぐために、ピンセットで神経にあまりにも多くのトラクションを適用することは避けてください。押す互いに離れる切り株、または全く再接続が発生しないことを保証するために、遠位神経の部分をカットし、削除する。 </li><li>挫滅損傷を実行した場合は、すべての軸索を切断する一定の圧力を用いて30秒間の神経を圧縮するデュモン＃45分の5鉗子を使用し、最初のクラッシュサイトに直交する第2の角度でこのクラッシュを繰り返します。そうでなければ怪我は動物の間一貫性がなくなります、30秒のクラッシュ時の圧力の可変量を適用することは避けてください。 </li></ol></li></ol> 4.閉会と回復<ol><li>下にある構造上の脂肪と筋肉を再配置します。 </li><li>切開のエッジを近似すると7.5ミリメートルの創傷クリップを使用して傷を閉じます。縫合糸または接着剤は、創傷閉鎖のためにも許容される。術後の鎮痛薬は、この時点で提供することができる。 </li><li>マウスの耳からテープを外します。イソフルランの流れをオフにして、マウスが1分30秒純酸素を呼吸することができます。 PL麻酔から回復する寝具で空のケージにマウスをエース。 </li><li>マウスが回復されると、顔面神経麻痺の確証的兆候がその挙動を調べる。ウィスカーが麻痺して戻って頬に向かって傾斜してなり、鼻がずれてしまう、との目は、空気のパフに応じて点滅しません。 </li><li>ハウス動物共同で、彼らが女性である場合は、手術後。彼らはより積極的であり、強制的に感染につながる、彼らのcagemateの創傷クリップを削除する傾向があるため、共同で雄マウスを収納しないでください。必要であれば、この時点では術後の鎮痛薬を提供します。 </li><li>全く感染または他の合併症は術後に発生していないことを確認するために操作した後、数日間一日一回のマウスを監視します。彼らは自分の上で落ちていない場合は、10日手術後 - 7創傷クリップを外します。 </li><li>目のまばたき反射が再いずれかになるまで、毎日の角膜合併症を防ぐために、罹患した眼に眼軟膏を潤滑適用カバーまたは安楽死まで、。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+mouse+sciatic+nerve+crush+and+subsequent+assessment+of+regeneration+by+whole+mount+muscle+analysis.">Reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis.</a> Journal of Visualized Experiments : JoVE. (60), (2012).</li><li>Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spared+nerve+injury+(SNI)+model+of+induced+mechanical+allodynia+in+mice.">The spared nerve injury (SNI) model of induced mechanical allodynia in mice.</a> Journal of Visualized Experiments : JoVE. (54), (2011).</li></ol>

The authors have nothing to disclose.

The critical step for this protocol is positioning the mouse properly before surgery is begun. If the mouse is not lying flat on its side, the ear is not taped at the correct angle, or the incision is made in an incorrect location, then finding the facial nerve becomes much more difficult. When this technique is mastered, surgeries will take only minutes per mouse. 
Either sutures, glue, or wound clips can be used to close the wound. Wound clips are preferred because of the small size of incision and rapidity with which they can be applied. We have determined that if wound clips are used and the mice are housed together, female mice fare better than male mice because males housed together often remove their cagemate&#8217;s wound clips, leading to infection of the surgical site.
Modifications can be made to the injury, such as changing the location along the nerve to induce injury. For example, the injury could be administered along one of the branches of the facial nerve28, or the skull could be opened to deliver an intracranial injury29. In addition, a variety of treatments can be implemented, such as applying drugs to the nerve stump14, injecting medicines systemically30, or even performing cell transfers31. Motoneuron diseases such as amyotrophic lateral sclerosis can also be studied using the facial nerve axotomy model32. Ongoing applications of this technique involve studying nerve reconnection strategies after transection and the application of pharmaceutical agents to accelerate nerve regeneration after injury. 
One of the most useful aspects of this surgery is that it is easy to confirm successful axotomy by monitoring the mouse&#8217;s vibrissae orientation and movements as well as the eye blink reflex. 
The paralysis does not affect the animal&#8217;s vital functions and overall is well tolerated in the mouse. 
A major advantage of the facial nerve injury model is that the isolated study of motoneurons is possible. However, because this is a cranial nerve injury, results found using this method may not translate to spinal, autonomic, or sensory nerve injury models. To study these other nerve injury types, a sciatic33 or tibial34 nerve injury model is recommended. The drawback of these models is that there is crosstalk from the contralateral neurons in the spinal cord that can confound results. There is no crosstalk between the facial motor nuclei, so the contralateral facial motor nucleus can serve as a paired internal control. The facial nerve axotomy model presents many advantages for studying nerve injury and regeneration.

多くの末梢神経損傷モデルが存在するが、運動ニューロンの研究のために際立っている1は、顔面神経軸索切断モデルです。また脳神経VIIとして知られている顔面神経は、橋に由来し、顔の表情1,2の筋肉を神経支配。この外科プロトコルでは、顔面神経は茎乳突孔からの出口で露出し、切断又は破砕のいずれかです。神経損傷の重症度を順次軸索の束を包む結合組織層である軸索、神経内膜、神経周膜、及び神経上膜の完全性に基づいて、けがを差別サンダーランド3分類、以下に分類することができる。挫滅損傷（軸索断裂）では、軸索が切断されているが、神経周膜と神経上膜は保持されます。無傷の神経鞘は軸索が4,5を再成長、その中の導管となるため、顔面神経挫滅からの完全な機能回復には約11日で発生する。上の一方では、カット損傷（神経断裂）に、軸索およびすべて3結合組織層が切断されており、全体の遠位神経は筋肉組織の神経支配を回復するために再成長しなければならない。神経上膜を外科的に再接続は、しばしば神経切断損傷を有するヒト患者において行われているが、回復の成果はめったに最適である。さらなる研究は、神経がその標的に再成長に失敗し、どの治療法が改善し、再生プロセスを加速するために使用することができるかを理解する必要がある。 顔面神経の軸索切断モデルを使用して神経損傷を研究する多くの利点がある。まず、顔面神経の軸索切断手順は、迅速、簡単、かつ再現性が高い。と顔の筋肉の結果として麻痺は重要な機能に影響を与えないとも動物によって許容されている。これは運動ニューロンが目の中で比較的均質な集団に存在するので、簡略化されて運動ニューロン細胞体を研究脳神経損傷モデルは、あるため、橋の電子顔面運動核。 7亜核が筋肉の特定のグループを支配するために、それぞれ固有のがあるので人口は、顔面運動核内の核内のパターンに基づいて異なるんので、軸索切断に応じて、核内の違いが結果の2,6,7に影響を与える可能性がある。 顔面神経損傷モデルの主な利点は、神経の神経支配は非常に対称的であり、顔面運動核8との間にクロストークがないのでunaxotomized側ペアリング内部対照として役立つことができることである。この外科的方法を使用することの別の利点は、直接CNSへの外傷または血液脳関門9の破壊がないことである。そのような過度の出血や感染症などの合併症は、この手順に稀である。 様々な分析は、神経損傷に対する生理学的応答を評価するために行うことができる。まばたき反射およびウィスカー活性の回復は、行動として使用することができ機能回復の10,11の尺度。鼻毛活動の録画は現在、顔面神経の神経支配12,13の回復を検出するための最も強力な方法である。安楽死の後、脳幹の組織学的分析は、顔面運動核内の運動ニューロン細胞体で行うことができる。顔面運動核の損傷2,6に対する応答の差検査を可能にする、特定の顔面筋にそれぞれ特定の七亜核に細分される。顔面運動ニューロンは、細胞の生存を定量化するためにカウントすることができ、または免疫組織化学は、バイオマーカーおよび特定の細胞集団14を識別するために使用することができる。顔面運動核を正確神 ​​経損傷15,16に対する細胞応答の分子解析のためのレーザーキャプチャーマイクロダイセクションを使用してすることができる。顔面神経の軸索切断の影響は、運動皮質17,18に分析することができる。また、神経はウォラー変性19またはを研究するために解剖することができます軸索再生20、および筋肉が神経筋接合部21を研究するために除去することができる。顔面神経の軸索切断はまた、22添付の中枢および末梢神経膠細胞を研究筋肉21、及び免疫系の応答23を標的とするために使用することができる。多くの顔面神経軸索切断モデル24を研究してなされたものであるが、神経損傷は、患者にとって重大な問題であり、現在の治療は、最適な結果を生み出すことができないので、末梢神経損傷のさらなる研究が必要である。このモデルは、神経損傷に対する生理的応答を調べ、神経再生治療の有効性を分析するための強力なツールである。

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
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			<td height="21" style="height:21px;width:381px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:256px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Stereo Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:176px;">M60</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Labeling tape</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">15-952</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5cm/5mm cutting edge</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">15003-08</td>
			<td>Sterilize before use</td>
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			<td height="42" style="height:42px;width:381px;">Dumont #5/45 Forceps - Standard tips/Angled 45&#176;/Dumoxel/11cm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">11251-35</td>
			<td>Sterilize before use</td>
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			<td height="21" style="height:21px;width:381px;">Michel Suture Clips - 7.5mm x 1.75 mm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12040-01</td>
			<td>Described as &#34;wound clip&#34; in protocol, sterilize before use</td>
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			<td height="21" style="height:21px;width:381px;">Hagenbarth Cross Action Wound Clip Applier 5&#34;</td>
			<td style="width:167px;">George Tiemann &#38; Co</td>
			<td style="width:176px;">160-910</td>
			<td>Used to apply wound clip, sterilize before use</td>
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			<td height="42" style="height:42px;width:381px;">Michel Suture Clip Applicator &#38; Remover - For 7.5 mm Clips</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12029-12</td>
			<td>Used to remove wound clip</td>
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			<td height="21" style="height:21px;width:381px;">0.9% Sodium Chloride Injection, USP</td>
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			<td height="21" style="height:21px;width:381px;">Betadine, 16 oz, with dispenser</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">19-027132</td>
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			<td height="21" style="height:21px;width:381px;">70% Ethanol</td>
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			<td height="21" style="height:21px;width:381px;">Glass Bead Sterilizer</td>
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</table>

facial nerve axotomy in mice: a model to study motoneuron response to injury

この外科プロトコルの目標は、茎乳突孔からの出口で、顔の筋肉組織を神経支配顔面神経を、露出させ、どちら末梢神経損傷を誘導するためにそれをカットまたは押しつぶすことです。この手術の利点は、このように、他の神経損傷モデルに比べて、比較的穏やかな手術結果をもたらし、その単純さ、再現性、およびその後の顔面神経麻痺から生体機能や移動度に対する効果の欠如である。脳神経損傷モデルを使用することの主な利点は、運動ニューロンは、運動ニューロン細胞体の研究を単純化する、橋顔面運動核における比較的均一な集団内に存在することである。なぜなら顔面神経の神経支配と顔面運動核の間のクロストークの欠如の対称的な性質のため、操作が対に内部対照としてunaxotomized側を一方的に行うことができる。様々な分析を評価するために術後に行うことができますこの記事の範囲を超えてその詳細は生理的反応、だ。例えば、筋機能の回復は、神経再生のための行動のマーカーとして役立つことができる、または運動ニューロンは、細胞の生存を測定するために定量化することができる。さらに、運動ニューロンを正確に分子解析のためのレーザーマイクロダイセクションを使用してキャプチャすることができる。顔面神経の軸索切断が最小侵襲性および忍容性が良好であるため、遺伝的に改変されたマウスの多種多様に利用することができる。また、この手術モデルは、末梢神経損傷の治療の有効性を分析するために使用することができる。顔面神経損傷は、運動ニューロンだけでなく、中枢および末梢神経膠微小環境の応答は、免疫系だけでなく、調査のための手段を提供し、筋肉組織を標的とする。顔面神経損傷モデルは、神経損傷および再生を研究するための強力なツールとなって広く受け入れられている末梢神経損傷モデルである。

顔面神経の軸索切断が行われた後、運動ニューロンの損失は、損傷の結果として起こる。損傷後の運動ニューロンの生存は、性別、手術時の動物の年齢、運動ニューロンのカウントが行われる時時点、及びモランとGraeberレビュー24と神野と山田レビュー22などの多くの変数が、運動ニューロンの生存データを要約の両方に依存する。一般的に、運動ニューロンの約86％が28日後に軸索切断の14,15,26で生き残る。運動ニューロンの損失の速度論はSerpe らに記載されている。2000 図1は、複数の遺伝的に改変されたマウスにおける運動ニューロンの生存の変化を示している。有意差は遺伝的変化は、ベースライン数に影響を与えないことを示す、制御側の顔面運動ニューロンの数において観察されない。 （2.0±84％; 図1A、D）野生型マウスにおける運動ニューロンの生存と比較して、有意な細胞損失がマウスモデルにおいて観察され筋萎縮性側索硬化症（SOD1 G93A; 68％±1、 図1B、E）、ならびに免疫不全組み換え活性化遺伝子2ノックアウトマウス27（RAG-2 - / - ; 図1C、F; 57％±2.5） 。 図2は 、顔面運動核に適用されるレーザーキャプチャーマイクロダイセクション法を示しています。全体顔面運動核（ 図2A-C）捕捉することができる、または亜核（ 図2D-F）を別々に回収することができる。より高い精度のために、運動ニューロンは、個々に捕捉することができ、残りの神経網の分析（ 図2G-I）のために収集することができる。 図3は、腹と腹外側亜核を比較する核内の試料から抽出したRNA物質の定量PCRの結果を示す。試験した4つの遺伝子、βIIチューブリン 、成長関連タンパク質-43（GAP-43）、hemopoietic-および神経-発現配列-1（HN1）、および脳由来神経栄養因子（BDNF）は、すべての神経再生応答に関連し、軸索切断16 ​​後二亜核およびそれらの遺伝子発現プロファイルとの間の興味深い違いがあるている。 <img alt="図1" src="/files/ftp_upload/52382/52382fig1highres.jpg" /> 図1.代表的な冠状顔面運動核切片をチオニンで染色し、28日目、顔面神経切断後に定量した。顔面運動核（A、D）WTから示され、（B、E）SOD1 G93A、および（C、F）RAG- 2 - / -マウス（制御側、軸索切断側）。スケールバー=120μmの。この図は、27から変更されている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52382/52382fig1large.jpg" target="_blank">目の拡大版を表示するには、こちらをクリックしてください</a>図である。 <img alt="図2" src="/files/ftp_upload/52382/52382fig2highres.jpg" /> 顔面運動核の図2.レーザーマイクロダイセクション。軸索切断顔面神経核の（A）チオニン染色切片、（B）部分的な軸索 ​​切断顔面神経核のレーザーマイクロダイセクション、およびレーザー顕微解剖組織の（C）は、コレクションを持つ。亜核のテンプレート（D）は、腹内側を識別し、レーザーマイクロダイセクション（E、F）のための顔の亜核の腹に、コンピュータ画面上に重ねた。顔面運動ニューロンは、レーザが（*運動ニューロン、G、Hを示す）、それらの可視核を有する形態学および核小体に基づいて、顕微解剖した矢印（G）で ​​示されるFMN細胞体断片は、レーザー別々顕微解剖およびFMN mRNAを除去するために廃棄しながら、た神経網s内のamples。すべてのFMNおよび細胞体の断片を回収した後、残りの顔面神経核組織は神経網のサンプル（I）としてレーザ顕微解剖した。スケールバー=100μmである。この図は、16から変更されている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52382/52382fig2large.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> <img alt="図3" src="/files/ftp_upload/52382/52382fig3highres.jpg" /> 図3.プロ再生及び生存促進mRNA発現腹内側で（VM）と ​​腹外側（VL）顔面神経軸索切断以下の顔面運動核内領域。に離断VMとVL顔の亜核の相対的なSEM±mRNA発現の平均パーセント未手術対照亜核（AD）。 mRNA発現の時間経過は、βIIチューブリンには傷害（0）、3、7、14、および28 DPO（Aを含まない</強い&gt;）、GAP-43（B）、HN1（C）、およびBDNF（D）。 ＃は、p &lt;0.05で、VMと比較して、VLの有意差を表す。この図は、16から変更されている。

Watch this Scientific Journal Video about Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury at JoVE.com

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

マウスにおける顔面神経軸索切断：傷害に運動ニューロン応答を研究するためのモデル

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

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Nanyang Technological University

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JoVE Journal

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12.8K ビュー.  Indiana University School of Medicine. We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques. 

ビデオ: Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

マウスにおける急性肺障害を誘発するために圧力制御された換気

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

医学

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

マウスで誘導機械的異痛症の倹約神経損傷（SNI）モデル

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

脛骨神経切断 - マウスにおける脱神経誘発性骨格筋萎縮のために標準化されたモデル

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

ポスト病変呼吸神経可塑性を研究するための子宮頸脊髄損傷のマウスモデル

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

三叉神経因性疼痛を研究するためにラットの眼窩下神経の慢性狭窄傷害（ION-CCI）

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

マウスにおける反復震とう頭部損傷モデル

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

マウスにおける組織変位ベースの脊柱挫傷モデルの検討

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...