この作品は、国防省 (DoD) 米国陸軍衛生研究資材コマンド (USAMRMC) 議会監督医学研究プログラム (CDMRP) (PR121604) と国立機関の関節筋骨格系からの補助金によって支えられました。や皮膚病 (NIAMS)、ユマ sankar さんに NIH R01 AR068332。

次の手順は、インディアナ大学学校の医学機関動物ケアおよび使用委員会 (IACUC) からの承認を行った。すべての生存手術は、NIH のガイドラインに従って滅菌条件の下で行われました。痛みや感染症のリスクは、適切な鎮痛剤と抗生物質の正常な結果を確保するために管理されていました。
1. 麻酔や準備
<ol><li>マウスの重量を量るし、ケタミン (100 mg/kg) とキシラジン (10 mg/kg) の投与の混合物を介しての腹腔内 (i. p.) ルートで麻酔すること。空の檻の中にマウスを置くし、それが完全に鎮静するまでそれを監視します。</li>
	<li>つま先ピンチ反射を使用して、マウスを鎮静することを確認します。その目を乾燥から保護するために眼軟膏を適用されます。</li>
	<li>右下肢から毛皮を削除します。ヨウ素系スクラブと 70% エタノールで手術部位を拭きます。手術部位、膝の中央から開始し、外側円形掃引をスクラブします。新鮮なスクラブ、70% エタノールで終わるこの 3 倍を繰り返します。</li>
	<li>即時術後疼痛管理のため皮下ブプレノルフィン塩酸塩鎮痛 (0.03 mg/kg) の術前投与量を管理します。</li>
	<li>滅菌手術用パッドで覆われて加熱パッドの上にマウスを置きます。</li>
</ol>2. 外科的アプローチ
注: 破断前に重量と落下高さ経験的決定してください特定のひずみ、年齢、手術前にマウスのセックスに。この手術は、10 週齢で C57BJ6 男性マウスに最適です。
<ol><li>その裏にマウスを置き、手術足の膝を屈曲します。メスの刃を使用して、膝関節の中央に 1.5 cm の切開を作る。</li>
	<li>横方向に鉗子を使用して大腿骨の遠位端を公開する膝蓋骨を置き換えません。大腿骨の近位端を逆行性に髄運河の長さの下の滑車溝の中心に 1.5 インチ長い 25 サージカル ステンレス製の注射針を挿入します。ピンの適切な配置を確保するため x 線を取る。 注: 針ガイドワイヤのパスを作成するのには、マウスの背側を終了する必要があります。</li>
	<li>大腿骨遠位部にハブを介して出入りマウスの背側に面取りをする針のシャフトを 4-36 ゲージ長いタングステン ガイドワイヤを渡します。</li>
	<li>ガイドワイヤの成功の配置、次の場所でリムとガイドワイヤを押しながらハブを注意深く引いて 25 ゲージの針を慎重に取り外します。X 線によるガイドワイヤーの配置を確認します。</li>
	<li>影響を与えるディスク (図 1 a) 上記 34.6 cm の高さから 391 g 重量を保持します。水平方向に 2 つの間で大腿骨の位置支持点、大腿骨の大腿骨、大腿骨領域サポート アンビル (図 1 b) の残りの部分、リムの側面には、荷重点 (図 1 を直面しているように).体重を落とすし、すぐに破壊された後のデバイスからマウスを慎重に取り外します。</li>
	<li>X 線による破壊の場所を確認します。</li>
	<li>骨折大腿骨を安定させるためにガイドワイヤ上 24 ゲージ ステンレス鋼管皮下に挿入します。 注: このアプリケーションのエントリ ポイントは、小さい直径の針を使用して生成された、いくつかの力を必要があります。この直径の違いは、大腿骨の近位端から 24 ゲージ棒の潜在的な移行を効果的に防止します。鈍のチューブは、大転子の皮質骨を満たしているように、挿入の深さは手動で感じることができます。</li>
	<li>ガイドワイヤを削除する前に x 線による骨折大腿骨の安定化と鉄筋の位置を確認します。</li>
	<li>ワイヤー カッターを用いた大腿骨の遠位端で余分なチューブをカットします。鉗子を使用して膝関節を脱臼しないように注意して、穏やかな下向きの力を適用する顆の表面の下で露出配管を埋めます。</li>
	<li>鉗子を用いた膝蓋骨の位置を変更します。5-0 吸収性縫合糸で切開部位を閉じます。</li>
</ol>3. 術後管理
<ol><li>術では、術後回復にそれらを支援するために i. p. ルートにマウス可能性があります最大 500 μ L の滅菌生理食塩水を介してを注入します。</li>
	<li>手術から目を覚ますまで、温水回復ベッド上の動物を監視します。一度、歩行、ケージに戻します。</li>
	<li>彼らは適切に治癒されており、可動性を取り戻すように手術後数日間マウスを密接に監視し続けます。ブプレノルフィン塩酸塩鎮痛 0.03 mg/kg 皮下すべて 6 h 術後、3 日間のおよび管理に応じてその後。彼らは手術後骨の治癒過程を阻害する示されている、非ステロイド性抗炎症薬 (NSAIDs) の使用を避けてください。</li>
</ol>

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この原稿の著者が明らかに何もありません。著者は、本稿で報告される調査で使用されるすべての材料へのフル アクセスに制限がない状態をさらに。

この手術の目的は、マウスで標準化された閉じた大腿骨骨折を生成することです。このモデルの主な利点は内部固定の髄内ロッドの湾曲を回避するため、破壊の発生後に行われる。おそらくこのプロトコルの最も重要な側面は、骨折の幾何学は、曲げ力や後肢の位置に依存して標準化された横骨折大腿骨骨幹の世代です。大腿骨の不適切な配置は、曲げモーメントの間に斜めのまたは粉砕骨?...

骨折などの筋骨格系に発生する最も一般的な傷害と $ 250 億アメリカ合衆国1で毎年を上回ると予想される治療費を含む多大な経済的負担に関連付けられて 2。骨折の大半は、何事もなく治癒、癒しは実質的なダウンタイムや生産性の損失に関連付けられています。全骨折の約 5-10% は、遅延癒しや非組合員、年齢や骨粗鬆症、糖尿病の3,4,5などの他の基になる慢性の健康状態のために します。現在効率的な骨の治癒を促進し、回復時間を短縮できる FDA 承認の薬理学的治療法がありません。
骨折治癒は種類の細胞の調整を含む複雑な非常にダイナミックなプロセスです。したがって、骨再生に関連する細胞・分子のイベントの包括的な理解は、このプロセスを加速させる治療標的の同定に重要です。他の人間の病気と同様に、非常に従順で再現可能な動物モデルの確立も骨の治癒の研究で非常に重要です。羊や豚などの大型動物骨改造プロパティとバイオメカニクス、人間に似ていますが高価、実質的な癒しの時間を必要とする、ない遺伝子操作6に簡単に従わないです。その一方で、ラットやマウスなどの小型の動物モデルは処理、メンテナンス、短い繁殖サイクル、および短い癒し時間7の低コスト性を含めて多くの利点を提供します。さらに、マウスのゲノムは、迅速な操作と遺伝的変異の生成を可能にする完全に、シーケンスです。したがって、マウスは人間の病気、怪我、研究および8を修復する強力なモデル システムです。人間では、骨粗鬆症や糖尿病の合併症は、治癒遅延の可能性を高めます。既存マウス モデルの数は骨の損傷に及ぼす骨粗鬆症や糖尿病などの合併症を研究利用可能癒し。骨粗鬆症患者治癒9破壊の後の段階で著しく減少した骨形成があります。卵巣摘出 (ovx) は、急速な骨量の減少と閉経後骨粗鬆症10,11にみられるような治癒遅延骨を示します。さらに、多くの種類のマウス ・ モデル私は、II 型糖尿病低骨マス表現型と模倣障害骨折治癒人間11に見られます。また、マウス骨折モデルはカルスで発生する複雑な生物学的過程を研究している多才なプラットホームとして役立ち、骨再生を加速する新規の治療戦略を探る。
骨の構造と代謝、骨骨折のままマウスとヒトに非常によく似たを治癒のプロセス全体の違いにもかかわらず軟骨と骨リモデリングに続いて膜内骨化の組み合わせを含みます。軟骨内骨化には、前駆細胞の肥大し、柔らかいカルスを生成する軟骨を石化する軟骨細胞に分化し、どこ破壊ギャップの周辺より機械的に安定した地域への採用が含まれます。前駆細胞の 2 番目の波は、カルスに潜入し、新しい骨マトリックス12,13,14,15を分泌する成熟した骨芽細胞に分化します。中膜内骨化、骨膜と骨の表面に前駆細胞は直接骨芽細胞の分泌の行列に区別して破壊ギャップ9,11,12 のブリッジを容易にします。 ,13。一緒に、軟骨と膜内の ossifications がさらに機械的負荷13,14 をサポートできる強力な二次骨を形成するため時間をかけて改造ハード カルスの開発につながる、15。健康な人間で癒しのプロセスは、マウス16で 35 日間と比較して、約 3 カ月かかります。
骨折治癒よく研究されているか開いているか閉じて手術モデル17を使用しています。批判的に大きさで分類された欠陥の生成などの外科的アプローチを開くまたは傷害の位置および粉砕骨折による偏差を軽減するジオメトリを標準化し、骨切りを完了します。閉鎖骨折と比較して治癒がよく遅れているので非組合員の背後にあるメカニズムを研究する優秀なモデルとしては、骨切り術。さらに、厳格な外部固定が再生は主に、膜内骨化の依存を意味、切り離した骨骨を安定させるために必要です。オープン手術アプローチが骨折した手足をもがれる軸と回転安定性を提供するためにロックの釘、ピン、クリップ、ロック プレートなどデバイスを使用します。ただし、このようなデバイスは高価であり、大きく手術18,19,20,21のより多くの時間を必要とします。その一方で、クローズド モデルは軟骨の治癒を促進するのに十分な不安定性を可能にする単純な髄内固定装置で安定しました。その結果、容易に閉じた破壊モデルは非組合員の条件を模倣しません。髄内ピン、釘、ネジ、圧縮などの内部固定技法は、安い、使いやすい、手術21,22,で23時間を最小限に抑えるため有利です。いくつかのケースで、破断前に髄内ピンを挿入が、湾曲や骨折大腿骨、変数カルスの大きさに貢献し、癒しの変位につながる髄内ピンの曲げ。破壊位置やジオメトリは、骨幹に重量をドロップする前記三点曲げ装置を使用して生成されたときクローズド モデルで標準化がより困難です。ただし、適切な技術と、この手術方法は迅速かつ一貫性のある結果を提供しています。また、閉鎖骨折モデルは高い力への影響や機械的ストレス22による骨折を研究する臨床的に関連性の高いツールとして提供しています。
この外科手術は、ラットおよびマウス22,24,25骨折大腿骨を安定させるために髄内ピンを使用して上記の方法から適応されました。まず、小径の髄内針はエントリのポイントを確立する intracondylar ノッチを通して挿入され、横重力依存性の 3 ポイントを使用して大腿骨骨幹骨折を生成する前にガイドワイヤーを導入曲げ装置です。閉じた大腿骨骨折の発生の成功、次のより大きな直径の髄内ロッドはガイドワイヤー骨折大腿骨を安定させるために組み込まれています。このメソッドは、治癒遅延、破壊時に髄内ピンの湾曲によって引き起こされる骨折後ロッドの配置により、負傷した大腿骨の再配置と最適化された安定化のため、危険を回避できます。

<table>
	<tbody>
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			<td>Oster Minimax Trimmer</td>
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			<td>POVIDONE-IODINE</td>
			<td>Thermo Fisher Scientific</td>
			<td>395516</td>
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			<td>OPHTHALMIC OINTMENT</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0490117</td>
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			<td>Styker T/Pump Warm Water Recirculator</td>
			<td>Kent Scientific Corporation</td>
			<td>TP-700</td>
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			<td>1ml Sub-Q Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>309597</td>
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			<td>ENCORE Sensi-Touch PF</td>
			<td>Moore Medical LLC</td>
			<td>30347</td>
			<td>Latex, powder-free surgical glove</td>
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			<td>PrecisionGlide 25G Hypodermic Needles</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-49</td>
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			<td>Ultra-High-Temperature Tungsten Wire,</td>
			<td>McMaster-Carr</td>
			<td>3775K37</td>
			<td>0.005&#34; Diameter, 1/16 lb. Spool, 380&#39; Long</td>
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			<td>304 stainless steel, 24G thin walled tubing</td>
			<td>Microgroup Inc</td>
			<td>304h24tw-5ft</td>
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			<td>#15 Scalpel Blades</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
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			<td>#10 Scalpel Blades</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
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			<td>Narrow Pattern Forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td>Serrated/Straight/12cm</td>
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			<td>Iris Forceps</td>
			<td>Fine Science Tools</td>
			<td>11066-07</td>
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			<td>Dissector Scissors</td>
			<td>Fine Science Tools</td>
			<td>14081-09</td>
			<td>Slim Blades/Angled to Side/Sharp-Sharp/10cm</td>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>ToughCut/Straight/Sharp-Sharp/11.5cm</td>
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			<td>Olsen-Hegar Needle Holder with Suture Cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td>Straight/Serrated/12cm/with Lock</td>
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			<td>Fine Science Tools</td>
			<td>13004-14</td>
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			<td>Tungsten Wire Cutter</td>
			<td>ACE Surgical Supply Co., Inc.</td>
			<td>08-051-90</td>
			<td>ACE #150 Wire Cutter, tungsten carbide tips</td>
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			<td>3-0 VICRYL Suture</td>
			<td>Ethicon Suture</td>
			<td>J423H</td>
			<td>3-0 VICRYL UNDYED 27&#34; FS-2 CUTTING</td>
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			<td>piXarray 100 Digital Specimen Radiography System</td>
			<td>Bioptics, Inc</td>
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			<td>Cabinet x-ray system</td>
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			<td>Einhorn 3-Point Bending Device</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom Built</td>
		</tr>
	</tbody>
</table>

the generation of closed femoral fractures in mice: a model to study bone healing

骨の骨折は、大幅に生活の質に影響を与えるだけでなく、患者に途方もない社会経済的負担を課する。効率的な骨の治癒を促進する治療戦略は存在しないと需要が高いです。骨折治癒の効果的かつ再現可能な動物モデルは、骨の再生に関連付けられている複雑な生物学的プロセスを理解する必要です。骨折治癒の多くの動物モデルは、長年にわたり; 生成されています。ただし、マウス骨折モデル最近骨の治癒を研究する強力なツールとして浮上しています。さまざまなオープンとクローズのモデルが開発されているが、生理学的に関連する方法で迅速かつ再現性のある結果を生成するための簡単な方法として際立っている閉じた大腿骨骨折モデル。この外科手術の目的は、マウスの一方的な閉鎖大腿骨骨折を生成し、髄内に鋼棒を挿入することによって大腿骨の骨折後の安定化を促進します。髄内ロッドの使用が骨組織で新たな不具合を生産やソフトの近くに損なうことがなく一貫した治癒転帰のための十分な安定化を提供しますが、釘やネジなどのデバイスより高い軸と回転安定性を提供組織。放射線イメージングを使用して、カルスの形成、骨性癒合および骨のカルスの後改造の進行を監視します。骨治癒転帰は通常治癒後の骨の強さに関連付けられているし、ねじり試験で計測。まだ、損傷修復に関連付けられている初期の細胞・分子のイベントについて骨再生の研究で重要です。髄内固定を持つマウスで閉じた大腿骨骨折モデルは骨骨折治癒研究・治癒を促進する治療戦略を評価するための魅力的なプラットフォームとして機能します。

手術の成功の実装は、放射線イメージングと監視されました。キーの手順には、髄内針、ガイドワイヤーの配置、横骨折大腿骨骨幹と (図 2 a私- 2Aiv) 髄内ロッドと適切な安定化の誘導の挿入が含まれます。破壊カルスの治癒の進行 (図 2 b) 術後 28 日を毎週放射線画像監視だった。日 10-16 後破壊で、...

Watch this Scientific Journal Video about The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing at JoVE.com

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

クローズド マウスの大腿骨骨折モデルは骨折治癒を勉強する強力なプラットフォームで骨の再生を加速する新しい治療戦略です。この外科手術の目的は、大腿骨を安定させるために髄内鋼棒を用いたマウスで一方的な閉鎖大腿骨骨折を生成します。

マウスで閉じた大腿骨骨折の生成: 骨の治癒を研究するためのモデル

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

the-generation-closed-femoral-fractures-mice-model-to-study-bone

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

SCIENTISTS IN ACTION

15.1K ビュー.  Indiana University School of Medicine. クローズド マウスの大腿骨骨折モデルは骨折治癒を勉強する強力なプラットフォームで骨の再生を加速する新しい治療戦略です。この外科手術の目的は、大腿骨を安定させるために髄内鋼棒を用いたマウスで一方的な閉鎖大腿骨骨折を生成します。

ビデオ: The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

生きたマウスにおける大腿骨髄吸引

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

マウスの簡単な重要なサイズの大腿欠陥モデル

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

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

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 either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

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 ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

プレート骨接合によって安定化マウスにおけるセグメント別大腿臨界サイズ欠陥モデルの確立

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

マウスにおける正常および不良骨治癒を分析するために大腿骨骨切り術の標準化された固定用髄内ロッキングネイル

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

歯髄の評価のための直接覆髄モデルの開発、創傷治癒およびマウスにおける修復象牙質形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

癒しの標準化された生体力学的条件下でマウスの軟骨破壊を分析する低侵襲モデル

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

マウスで横方向の大動脈狭窄を誘導するために閉じて胸モデル

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...