この作品は、CIHR筋研究パートナーシップ（ - ; JAEBへ90959 JNM）からの補助金によって支えられて。

前にこのモデルを使用することに、研究者は彼らの機関の動物使用統治体からの外科的プロトコルの承認を受けている必要があります。モデルは、研究倫理委員会、ハミルトン·ヘルス·サイエンス社、マクマスター大学（AUP＃10-04-24）によって承認されており、動物のケアカナダ評議会の勧告に厳密に従って実施される。 1。マウス準備<ol><li>マウス重量を量る。 5イソフルラン％または2％ハロタンで麻酔を誘導する。使用される回路は、外科医を保護するために麻酔の掃気適切性を確保する必要があります。 2-3分後、動物の呼吸が遅くなります。まばたき反射が不在であることを確認し、手術麻酔を（マウスでも応答すなわちなし ）を確認するために足ですだれスペースをつまむ。手術中に角膜の乾燥を防ぐために、眼に眼用潤滑剤を適用する。 </li><li>坐骨からラテラル太ももや臀部を剃る膝にノッチ及びproviodineで消毒。剃毛は、手術野の適切な可視化を確実にするために、神経解剖と離断との干渉を最小限にするために切開毛のサイトは自由を維持します。優れたと大腿骨の後方にある坐骨ノッチは、触診によって識別することができます。 </li></ol> 2。手術手技<ol><li> 2％（ハロタン1％）に吸入イソフルランを軽減し、操作や解剖顕微鏡下でその側（上向きに手術のために意図された側）、上にマウスを置きます。 3.5X倍率が成人（20〜25 g）のマウスには十分であるため、別の方法として手術は外科ルーペを用いて行うことができる。 </li><li>滅菌手袋を着用。触診で坐骨ノッチを識別します。メスを使用して、坐骨ノッチから膝への横方向の大腿（約1cm）の皮膚を切開。 </li><li>優しく肌を広げた。大腿二頭筋を特定し、これは、横方向の大腿部の平らな表面的な筋肉です。すぐに皮膚の下。細かいハサミを使用して、筋線維に沿って大腿二頭筋を分割し、坐骨神経とその枝を公開するために、スプリングリトラクタと開いたまま。 </li><li>大腿二頭筋に直ちに深い坐骨神経を識別します。それは、その特徴的な光沢のある白い色で識別され、直径が約0.8mmですることができる。これは、膝窩のレベルで、脛骨腓骨と腓腹神経に分岐、坐骨ノッチから膝まで実行されます。 </li><li>優しく超微細鉗子と春microdissectingハサミで腓骨と腓腹神経の枝から脛骨を分離。脛骨神経は、最大の枝であり、通常の中心です。それは枝を分離しながら神経をつぶすしないことが重要です。超微細鉗子でただ外側adventicial層の上に神経を保持し、かつ神経たるみを（教えていない）維持、神経挫滅とトラクションの傷害を避けることができます。 </li><li>完全かつ持続的denervati用上で、慎重に膝血管を避け、遠できるだけはさみmicrodissectingと脛骨神経を切った。また、2〜4週間の予想を完全再支配との一時的な除神経のために、脛骨神経は単純に15秒の代わりに離断のための超微細鉗子で粉砕することができます。 （末梢神経損傷後の再成長と対象筋肉をreinnervateます。） </li><li>完全な除神経が必要な場合は、腓腹筋の異常な再神経支配を防止するために10-0ナイロンおよび再近似5-0 VICRYLと大腿二頭筋と大腿二頭筋の前面に切開脛骨神経の端部を縫合しヒラメ筋。 </li><li>実行中の5-0 VICRYL縫合糸で皮膚を閉じます。 </li></ol> 3。手術ケアを投稿<ol><li>吸入麻酔薬をオフにしますが、酸素の流れを維持します。皮下buprinorphine（または代替）鎮痛剤を投与する。 </li><li>ないクリーンなケージにマウスを移しません寝具麻酔から目覚めた。 ambulatingまでケージ内および直接観察下温暖毛布の上においてください。 </li><li>十分柔らかい寝具柔らかい底のケージ内の転送と家（ではない線）。 </li><li>手術の傷や褥瘡ヒール潰瘍や噛むの証拠の開発のための足の状態を毎日手術肢を点検。軽微なトラブルは、proviodineなどの局所抗生物質や防腐剤を使用して管理することができます。動物の安楽死を必要とするエンドポイントインジケータは減量、貧しい自己ケアのエビデンス（フリルファー）、および猫背姿勢です。また、主要な傷の中断または局所抗生物質で1〜2週間で治癒や痛みを持っているようには見えない潰瘍と動物を犠牲にすべきである。 </li></ol> 4。除神経腓腹筋とヒラメ筋の収穫<ol><li>希望術後時点で、CO 2過剰摂取でマウスと犠牲を量る。 </li><li>両方の内側側面を剃る手術および対コントロールの足とアルコールできれい。操作や解剖顕微鏡下にマウスを置き、あるいは拡大するため手術視野ルーペを使用しています。 </li><li>手術肢で、足首周り足首から膝にメスと円周方向と内側カーフスキンを切開。優しく筋肉オフと近位大腿部に向かって鉗子で皮膚を引っ張る。これは、脚のすべての筋肉を公開します。脚の後部側面に膝から足首まで実行され、皮膚の下にすぐにあるふくらはぎの筋肉である腓腹筋を識別。膝の内側側面に腓腹筋の近位、大腿二頭筋の遠位挿入を識別します。その遠位挿入で大腿二頭筋は薄く、フィルム状に表示され、腓腹筋の最も近位部分に重なる。はさみや平滑末端郭清を使用した、優しくガスから大腿二頭筋の遠位挿入を分離trocnemius筋肉。 </li><li>踵には、その遠位挿入では、腓腹筋はアキレス腱に先細り。白と筋骨たくましい現れアキレス腱を識別。ホールドまたは腓腹筋を鎮圧しないように注意しながら、ピンセットでアキレス腱を保持し、ハサミを使って踵挿入からアキレス腱を分割。 </li><li>まだ腱を保持、そっと膝を原点に向かって遠位挿入（ヒラメは別途収穫することができる）から、深いヒラメ（深い赤）から腓腹筋（淡赤色）を持ち上げます。 </li><li>はさみを使って内側と外側大腿chondylesから腓腹筋の起源を割って脚オフ腓腹筋を解剖。筋肉に非常に穏やかなトラクションは、このプロセスを容易にします。筋肉を粉砕しないように注意してください。 </li><li>ヒラメは今直ちに腓腹筋のサイトの下に、はっきりと見えるようになります。そのにアキレス腱への挿入からヒラメを持ち上げたり後部ふくらはぎにイギン。ヒラメが誤っ腓腹筋で発生した場合、優しく収穫標本から分離。腓腹筋（光赤）とヒラメ（暗赤色）は、それらの色の違いによる収穫標本で容易に識別残る。 </li><li>精密スケールで別々に筋肉を量る。 </li><li>液体窒素で冷却したイソペンタン中で10％ホルマリン、または凍結固定で組織学のために液体窒素中でスナップ凍結用の半分（抽出、その後のタンパク質および/ ​​またはRNAの場合）、ハーフ（ すなわち形態計測評価、免疫組織学）のいずれか固定、垂直方向に筋肉を分割、必要に応じて。 </li><li>制御腓腹筋とヒラメ筋を収穫するコントロールで上記のステップ4.3、未作動、側から繰り返します。 </li></ol>

<ol>
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利害の衝突は宣言されていません。

除神経誘発性骨格筋萎縮の脛骨神経切断モデルは、ラットで一般的に採用し、十分に検証されたモデルです。私たちは、捜査官は、遺伝子改変マウスの存在を活用し、筋肉量7,8の規制に重要なタンパク質が存在しない場合に、生体内での筋萎縮の過程を研究することができ、マウスで使用するため、このモデルを適応している。腓腹筋とヒラメ筋、このモデルでは除神経の両?...

外傷性末梢神経損傷、疾患や薬理学的介入のために骨格筋の除神経、筋肉の自発的収縮機能の即時損失結果。筋肉が付随して萎縮し始め、タイムリーな、上質な再支配が1,2が発生した場合、この萎縮が可逆的である。再支配が存在しない場合には、筋線維の萎縮が進行し、筋肉における不可逆生物学的変化は、筋線維と筋線維の死で発生。ここでは、マウスの脛骨神経切断モデル、除神経誘発性骨格筋萎縮や線維症のモデルを示しています。このモデルは、腓腹筋とヒラメ筋のin vivoでの筋萎縮の根底にある生理学的、細胞および分子の生物学的メカニズムを研究する科学者が可能になります。歴史的にノックアウト、特にトランスジェニックマウスラインにこのモデルのラット、より最近のアプリケーションで主に使用されているが、研究者が自分の役割を評価することができますin vivoでの誘導、開発と保守、または代わりの解像度、筋萎縮と線維症への関心の特定のタンパク質（秒）。 脛骨神経は、げっ歯類の後肢における混合モータ感覚末梢神経であり、坐骨神経の三端子の枝の一つです。脛骨神経の横断は、腓腹筋、ヒラメ筋と足底の筋肉を（および後脛骨筋、指屈筋長母と屈筋hallicus長母を含め、足の3小深い屈筋）denervates、及びラット3,4でよく標準化され、検証モデルである。腓腹筋とヒラメ筋を容易にシリアル時点で切開することができる脛骨神経切断、固定され、処理された筋肉組織学および筋肉繊維形態計測の評価のために、又はフラッシュは、例えば研究するための筋RNAおよびタンパク質の抽出のために凍結し、ポスト筋萎縮を調節する細胞内シグナル伝達ネットワーク。 GAstrocnemius筋は混合繊維の種類の筋肉（I型とII型、主にII型が）で、ヒラメ筋、それによって評価5,6のために高速と低速の両方筋筋を提供する、タイプI線維の大部分で構成されています。脛骨神経切断モデルは、短期（日）7と長期（週か月まで）4,8両方で除神経誘発性筋萎縮の過程を研究するのに適しています。 坐骨神経切断モデル（一般的なげっ歯類に使用される除神経誘発性筋萎縮の第二モデル）とは対照的に、脛骨神経切断は、より魅力的なモデル作り、動物の少ない合併症を誘発する。脛骨神経の横断は、坐骨神経の腓骨と腓腹神経の枝はそのまま残り、一方坐骨神経の横断は、このように保存し、2を歩き回るために動物の能力を損なう、脚（ひざ下）と足のすべての筋肉をdenervates彼らのターゲットの筋肉と感覚領土。マウスは足底フレックスにできないか、足を反転しますが、簡単に歩き回るすることができ、重量が著しくモデルの罹患率を減少させる、両方の後肢に均等に負担する。歩行パターンを評価する歩行分析研究は、脛骨と坐骨神経傷害後のラットで行われ、そのフットプリントを示し、体重負荷の方が脛骨傷害9,10で保存されています。研究デザイン3を必要とする場合、また、脛骨神経切断モデルにおいて、腓骨神経は、後述時点で動員することができ、遅延された神経再生の供給源として転送される。これとは対照的に、坐骨神経離断モデルにおける遅延再支配は非常に大幅にモデルの技術的な難しさを増加させ、熟練した外科医にその使用を制限し、坐骨神経の赤字に神経移植の使用を必要とする。 しばらく脛骨神経切断モデルRそれが神経支配脛骨神経とふくらはぎの筋肉の両方の動物の手術における無菌手術技術と演算子の親しみを、equires外科医ではない、または高度に動物の手術の経験が個人は、容易にこのモデルをマスターできるように、操作のために容易にアクセスし、識別可能。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>10-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>2850G</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Vicryl suture</td>
			<td>Ethicon</td>
			<td>J553G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Spring microdissecting scissors</td>
			<td>Fine Surgical Tools</td>
			<td>15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Fine Surgical Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Non locking micro needle holder (driver)</td>
			<td>Fine Surgical Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring retractor</td>
			<td>Fine Surgical Tools</td>
			<td>17000-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice

脛骨神経切断モデルは、げっ歯類における除神経誘発性骨格筋萎縮の、良好な忍容性を検証し、再現可能なモデルです。独自に開発し、その大きなサイズに起因したラットで広く使用されていますが、マウスで脛骨神経は、無傷の坐骨神経の腓骨と腓腹神経の枝を残して、それによって維持し、それは簡単にクラッシュまたは離断のいずれかで操作することができる十分な大きさその標的筋肉。したがって、このモデルでは、坐骨神経切断モデル未満の罹患率と歩行の障害を誘発することの利点を提供し、また、研究者が遺伝子組み換えマウスでは筋萎縮の過程を規制する、生理的細胞および分子の生物学的メカニズムを研究することができます。脛骨神経は、腓腹筋、ヒラメ筋と足底の筋肉を供給しているので、その離断は、速筋のII型繊維および/または遅筋型から成る除神経骨格筋の研究を可能にし、私繊維。ここでは、C57Black6マウスにおける脛骨神経切断モデルを示しています。我々は、パラフィン包埋組織切片で筋量及びファイバ型特定の断面積を測定することによって、1,2において、代表的な筋肉のように、腓腹筋の萎縮を評価し、4週後の除神経は、速筋ミオシンための免疫染色。

<html>脛骨神経切断は、ふくらはぎの腓腹筋、ヒラメ筋と足底の筋肉をdenervates。ここでは、代表的な筋肉として、腓腹筋における萎縮の開発を評価する。腓腹筋は1,2、または4週間除神経2-3ヶ月齢C57Black 6マウス（ジャクソン研究所）から採取した。タイプII速筋の筋線維（ 図2）の断面積が行うように、筋肉の量は徐々に時間をかけて、（ 図1）減少する。腓腹筋は、混...

Watch this Scientific Journal Video about Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice at JoVE.com

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

脛骨神経切断モデルは、骨格筋萎縮の、良好な忍容性を検証し、再現可能なモデルです。モデル外科用プロトコルはC57Black6マウスにおいて説明され、示されている。

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. ...

tibial-nerve-transection-standardized-model-for-denervation-induced

Research

JoVE Journal

Medicine

24.3K ビュー.  St Michaels Hospital. 脛骨神経切断モデルは、骨格筋萎縮の、良好な忍容性を検証し、再現可能なモデルです。モデル外科用プロトコルはC57Black6マウスにおいて説明され、示されている。 

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

単離骨格筋のミオ - 機械的分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

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 ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

プロテオミクスから骨格筋の性別二型

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

マウスにおける神経因性疼痛の坐骨神経細胞浸潤モデル

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

変更されたベルイストローム法を用いたヒト骨格筋生検手順

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

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 ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人間<em&gt;外側広筋</emワイル・ブレークスリーConchotomeを使用&gt;骨格筋生検

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

豚脛骨移植外科的血管新生: 新しい大型動物の骨血管複合移植モデル

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...