著者の一人（ディオゴカザル）は、電子Tecnologia、ポルトガル電子FundaçãoパラACiênciaMINISTERIOダSaúde、Fundaçãoカルースト・グルベンキアン、Fundaçãoシャンパリモーが主催している高度な医学教育のためのプログラムからの助成金を受けました。 著者らは、ビデオを撮影し、編集中の氏アルベルト・セヴェリーノの技術的なサポートに感謝したいと思います。著者らはまた、本論文で動物の標本を調製する際に彼らの助けのための氏オクタビオChaveiro氏、マルコ・コスタ氏とカルロス・ロペスに感謝しています。 最後に、著者らは、動物の獲得と維持に関連するすべての物流の面で彼女の助けのために氏Gracindaメネゼスに感謝したいと思います。

動物を対象とするすべての手順は、制度的動物実験委員会および倫理委員会ノヴァ大学医学部、リスボン、ポルトガル（08/2012 / CEFCM）によって承認されました。 1.手術手順セットアップの注意事項<ol><li> 250〜350グラムの体重の成人のウィスターラットを使用してください。 </li><li> 7日手術前12時間の明暗サイクルで食物および水を随意にラットを保管してください。 </li><li>必要な麻酔薬の量を決定するために、ラットを計量。 </li><li>手術前に、すべての手術器具をオートクレーブ。 </li><li>手続きに必要なレイアウトは、すべての外科用品や楽器（材料の表を参照してください）。 </li><li>従来と顕微の機器を使用して、手術用顕微鏡下手術を行います。 </li><li>恒温ブランケット、直腸プローブ、および加熱ランプを配置します。 </li><li>水浴中で0.9％の生理食塩水を含有する1 20ミリリットル滅菌バイアルを置きに温め37ºC。 </li><li>アルコール溶液で動作設定の全ての表面を消毒するために滅菌手袋を着用してください。手袋を外します。 </li><li>スクラブキャップとマスクを配置します。 </li><li>水と石鹸で手を消毒し、滅菌手袋の別のペアを着用してください。 </li><li>無菌手術用ガウンを着用してください。 </li></ol> 2.麻酔と皮膚の準備注：滅菌ガウンと手袋を着用しているとして、次の4つの手順でアシスタントの助けを持っています。 <ol><li>腹腔内投与ケタミンとジアゼパムの混合物でラットを麻酔。投与量を5mg / kgのケタミンおよび0.25ミリグラム/キログラムジアゼパムです。つま先のピンチによって、全手順8,14,15,32を通じて呼吸速度の遵守により麻酔深度を判断します。 </li><li>角膜剥離を回避するために、目の前表面の上に眼科用ゲルを適用します。 </li><li>脱毛クリームと腹部の腹側表面に毛を削除します。ヘアremov後アルは、温かい生理食塩水で脱毛クリームを削除します。 </li><li>手術部位の上にアルコール溶液のかなりの量をスプレーします。手術部位で製品を残し、それを拭かないでください。少なくとも15秒待ってください。アプリケーションを3回繰り返します。手術を続行する前に少なくとも2分の接触時間を残します。他の研究ユニットは、手術部位感染を予防するために、他のプロトコルを使用します。 </li><li>滅菌手袋を着用して、ラットの両側に2外科用ドレープを配置します。 </li></ol> 3.ドナー部位の外科的処置<ol><li> 長さが約5cm 3センチ幅の範囲上腹部フラップの境界を設定します。 <ol><li>外科的皮膚マーカーを使用して、ラットの腹部の腹側表面上に正中線をマークするために、恥骨結合に胸骨の剣状突起から線を引きます。 </li><li>ラットの左側に、外科用皮膚マーカーを使用して、最初の行への2つの垂直な線を描く：オンeは胸郭のすぐ尾交差し、別の1、後者と（ 図3と図 6）鼠径部の倍だけ頭蓋に平行。 </li><li>マークライン正中線に平行と離れてそれから約3センチメートルと外科的皮膚マーカーで横切開。 </li></ol></li><li> フラップ収穫 <ol><li> 皮筋層の層に達するまで、15番メスの刃で皮膚を切開。 </li><li> 皮筋層面に深く、筋肉の筋膜に達するまで電気メスで切開します。 </li><li>横ために内側からフラップを上げ、頭蓋から尾側に、フラップの椎弓根を露出させます。 </li><li>穿孔血管が深い筋層から上がってくるとフラップの深い面に入る慎重を結紮し、分割します。 </li><li>フラップの尾側の側面に開創器を置き、優しくいじめによって慎重フラップの椎弓根を分析離れ緩い周囲の組織（ 図7）。 </li><li>結紮および外側大腿回旋動脈を分割し、合字のための9/0ナイロンを使用して静脈。 </li><li>大腿動脈と静脈を分離します。存在する場合、（9/0ナイロンを使用して）連結し、隣接する筋肉にこれらの血管の枝を分けます。 </li><li>まず、大腿静脈の近位側面をクランプするために二重の血管クランプを使用します。続いて、その遠位面をクランプします。その後、大腿動脈の遠位面と最終的にその近位側面をクランプします。 </li><li>大腿動脈の遠位面と最終的にその近位側面を固定します。 </li><li>浅腹壁静脈内の単一の血管クランプと浅腹壁動脈内の別の1を置きます。それぞれ、その起源と終了時に浅腹壁動脈と静脈をカットするストレート顕微はさみのペアを使用してください。 </li><li>多量のヘパリン処理通常の生理食塩水10 IU / mlのこれらの血管の内腔を洗浄、血液や破片がないまで、船舶の内腔33の内側に見られています。 </li><li>血管セクションのサイトに近い外膜のカフを引き、トリム。 </li><li>アディソンの鉗子（ 図8）を使用して、首に心窩部フラップを転送します。 </li><li>表皮下でドナー部位を閉じ5/0吸収性ステッチを中断。 </li><li>中断された5/0ナイロンステッチで皮膚を閉じます。 </li></ol></li></ol> 4.受信者のサイトの外科的処置<ol><li> 受信者のサイト船舶の暴露 <ol><li>外科的皮膚マーカーを使用して、左胸鎖乳突筋（SCM）筋の内側縁の上に線を引きます。 </li><li>外科的皮膚マーカーを使用して、左鎖骨のすぐ頭蓋と並列別の線を引きます。この2行は、左側胸鎖関節に収束しなければなりません。 </li><li> 15番手術用メスの刃を使用して皮膚を切開。 </li><li> subcutaneouを切断するために電気メスを使用して、sの組織。 </li><li> SCMの筋肉への横方向の外頚静脈を骸骨にするために解剖ハサミを使用してください。 </li><li>この（ 図9）に外頸静の支流を単離し、ライゲーション。 </li><li>ちょうど9/0ナイロン縫合糸と下顎の下に外頸静脈を結紮。 </li><li>後者のライゲーションの下に単一の静脈クランプを置き、ストレート顕微はさみのペアを使用して静脈外頸静を切りました。 </li><li> 10 IU / mlの濃度でヘパリン添加生理食塩水の静脈の内腔を洗浄します。 </li><li> SCM筋の内側縁を分離するため、頸動脈及び迷走神経（ 図10）をさらす、横方向にこの筋肉を撤回。 </li><li>電気メスを使用して、SCM筋の中部3分の1で横切開を行います。 </li><li> SCMの筋肉とストラップの筋肉の深い表面との間のリトラクターを置きます。 </li><li>頸動脈から迷走神経を離れていじめます動脈、これらの構造に損傷を与えないように注意しながら。 </li></ol></li><li> 血管吻合 <ol><li>頸動脈内に二重動脈クランプの位置を調整します。 </li><li>頸動脈の外側面に9/0ナイロンステッチを配置し、血管壁のこの部分を引っ張って、このステッチを使用しています。 </li><li>血管壁のこの領域に開口部を生成するためにまっすぐ顕微はさみのペアを使用してください。 </li><li>使い方は10/0ナイロン縫合糸は、フラップの浅腹壁動脈と最近作成した頸動脈開口部のレベルでの頸動脈の間termino横吻合を行う中断しました。 </li><li>外頸静脈の近位断端と浅腹壁静脈に接近し、これら二つの静脈の口径を検査します。 <ol><li>サイズの不一致が緩やかにわずかである場合は、拡張鉗子を使用して、浅腹壁静脈の切断端の内腔を拡張させます。 </li><li>口径の差は非常にPRONである場合ouncedは、鉗子の拡張に加えて、30〜45°の角度で浅腹壁静脈の端部を面取り。 </li><li> 11/0ナイロン縫合糸を中断使用して、静脈吻合を実行します。 </li></ol></li><li>フラップの船の上に配置された単一のクランプを外します。 </li><li>大腿静脈内に配置さダブルクランプを取り外します。 </li><li>大腿動脈に配置された二重クランプを取り外します。 </li></ol></li><li> 吻合の開存性と能力を評価します <ol><li>フラップの動脈と静脈が完全に拡張されており、有意な出血は血管クランプ（ 図11）を除去の3分後に観察されないかどうかを確認します。 <ol><li>出血は、この期間中に存在する場合は、吻合の上に湿った生理食塩水ガーゼを置き、穏やかな圧力を適用します。 </li><li>吻合からの出血が3分後に停止しない場合、必要に応じてナイロン、血管クランプ配置後、縫合糸を中断11/0追加追加します。 </li></ol></li><li>フラップ首の血管に接続し、温生理食塩水で湿らせたガーゼでラップして10分を待ちます。 </li><li>フラップの灌流と首の傷の止血を評価します。出血、血栓症や過度の牽引の兆候吻合を点検。 </li><li> 5/0皮内結節縫合で始まる受信者サイトでフラップを固定します。 </li><li>ナイロン縫合糸を中断5/0で皮膚（ 図12）を閉じます。 </li></ol></li></ol> 5.術後ケア<ol><li>右側臥位でその個々のケージの内側に回復するために、ラットを残します。下の低に設定された電熱パッドを配置することによって、ケージを暖かく保ちます。ケージや温熱療法を避けるために、電熱パッドとの間の光布を置きます。 </li><li>それは胸骨横臥を再開し、歩行できるようになるまで、連続して反対側の側臥位に5分ごとにそれを回す動物をご覧ください。 </li><li>ハウストンを除去するまでの個別ラット彼の外科ステッチの外科的処置後2週間。 </li><li>術後鎮痛のために、手術後3日間抗炎症薬には1mg / kgを皮下に一日一回を与えます。 </li></ol> 6.フラップ評価<ol><li>ラットの頭の上に食品の御馳走を提示し、目視によりフラップの生存率を評価。 </li><li>暴露前の手順を使用して不足している場合は、フラップを調べながらアシスタントは、ラットの肩甲骨間領域にわたって優しいタッチを適用しています。 </li><li>トルヒーヨらによって詳細に説明するように定量的に、創傷裂開、フラップ表皮、充血、うっ血および/または壊死の領域を評価するために、デジタル写真とImageJソフトウェアを使用してください。 15。 </li></ol>

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N., Browne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+flap+design+of+the+epigastric+artery+flap+in+rats--a+new+experimental+flap+model.">Modification in flap design of the epigastric artery flap in rats--a new experimental flap model.</a> Ann Plast Surg. 39 (5), 500-504 (1997).</li><li>Strauch, B., Murray, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+composite+graft+with+immediate+suture+anastomosis+of+its+vascular+pedicle+measuring+less+than+1+mm.+in+external+diameter+using+microsurgical+techniques.">Transfer of composite graft with immediate suture anastomosis of its vascular pedicle measuring less than 1 mm. in external diameter using microsurgical techniques.</a> Plast Reconstr Surg. 40 (4), 325-329 (1967).</li><li>Green, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the Rat. First edn. , 124-153 (1968).</li><li>Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the rat. , (1959).</li><li>Langworthy, O. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+study+of+the+panniculus+carnosus+and+its+genetical+relationship+to+the+pectoral+musculature+in+rodents.">A morphological study of the panniculus carnosus and its genetical relationship to the pectoral musculature in rodents.</a> Am J Anat. 35 (2), 283-302 (1925).</li><li>Popesko, P., Ratjov&#225;, V., Hor&#225;k, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Colour Atlas of the Anatomy of Small Laboratory Animals. 2, 13-104 (1992).</li><li>Brown, S. H., Banuelos, K., Ward, S. R., Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+morphological+assessment+of+rat+abdominal+wall+muscles:+comparison+for+use+as+a+human+model.">Architectural and morphological assessment of rat abdominal wall muscles: comparison for use as a human model.</a> J Anat. 217 (3), 196-202 (2010).</li><li>Harder, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+tissue+injury+in+the+dorsal+skinfold+chamber+of+the+mouse:+a+skin+flap+model+to+investigate+acute+persistent+ischemia.">Ischemic tissue injury in the dorsal skinfold chamber of the mouse: a skin flap model to investigate acute persistent ischemia.</a> J Vis Exp. (93), e51900 (2014).</li><li>Cox, G. W., Runnels, S., Hsu, H. S., Das, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+heparinised+saline+irrigation+solutions+in+a+model+of+microvascular+thrombosis.">A comparison of heparinised saline irrigation solutions in a model of microvascular thrombosis.</a> Br J Plast Surg. 45 (5), 345-348 (1992).</li><li>Gurunluoglu, R., Siemionow, M. Z., Siemionow, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastic and Reconstructive Surgery: Experimental models and research designs. , 53-62 (2015).</li><li>Nasir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plast Reconstr Surg. , 227-236 (2015).</li><li>Parsa, F. D., Spira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+anastomotic+techniques+in+the+experimental+transfer+of+free+skin+flaps.">Evaluation of anastomotic techniques in the experimental transfer of free skin flaps.</a> Plast Reconstr Surg. 63 (5), 696-699 (1979).</li><li>Dunn, R. M., Huff, W., Mancoll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rat+Rectus+Abdominis+Myocutaneous+Flap:+A+True+Myocutaneous+Flap+Model.">The Rat Rectus Abdominis Myocutaneous Flap: A True Myocutaneous Flap Model.</a> Ann Plast Surg. 31 (4), 352-357 (1993).</li><li>&#214;zkan, &. #. 2. 1. 4. ;., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+flap+model+in+rats:+iliac+osteomusculocutaneous+flap.">A new flap model in rats: iliac osteomusculocutaneous flap.</a> Ann Plast Surg. 47 (2), 161-167 (2001).</li><li>Ozkan, O., Koshima, I., Gonda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+supermicrosurgical+flap+model+in+the+rat:+a+free+true+abdominal+perforator+flap+with+a+short+pedicle.">A supermicrosurgical flap model in the rat: a free true abdominal perforator flap with a short pedicle.</a> Plast Reconstr Surg. 117 (2), 479-485 (2006).</li><li>Dorsett-Martin, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+models+of+skin+wound+healing:+a+review.">Rat models of skin wound healing: a review.</a> Wound Repair Regen. 12 (6), 591-599 (2004).</li><li>Ghali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+chronic+wound+infections+with+genetically+modified+free+flaps.">Treating chronic wound infections with genetically modified free flaps.</a> Plast Reconstr Surg. 123 (4), 1157-1168 (2009).</li></ol>

The authors have nothing to disclose.

The most important aspect to obtain consistent flap survival is paying attention to detail in various steps of the microsurgical technique. For example, to obtain good visualization of the vessels, of the surgical instruments and of the fine suture lines, it is very helpful to place underneath the vessels, a sterilized colored plastic background. As many researchers, we prefer to use sterilized fragments of yellow or green balloons (Figures 7 and 11). This background provides the additional advantage of minimizing adherence of suture lines to the adjacent structures, which sometimes leads to the need of pulling the suture line with too much tension, which may in turn lead to vascular tearing. Finally, the use of a background has the additional advantage of decreasing the probability of inadvertently dragging potential thrombogenic tissue debris to the anastomosis site.
Considering that the flap's vessels are very fine and fragile, it is important not to pinch the entire width of the vessels, in order to avoid intimal lesion that, in turn, will lead to intravascular thrombosis and flap failure. To prevent inadvertent injury to both the flap's vessels and to the recipient site's vessels, it is safer to liberally ligate and divide neighboring tributaries, which will allow an easier manipulation of these vessels.
Before starting the anastomoses, it is vital to place the vessels in their definitive position, striving to prevent vascular kinking or torsion of the flap's pedicle. Given the small caliber and delicate consistency of the vessels, these are often difficult to exclude unequivocally. One helpful trick is to secure the flap in its final position with 3 stitches placed away from the site of the anastomoses. Next, if in doubt, temporarily open the vascular clamps placed at the flap's pedicle, and fill the vessels' lumen with heparinized normal saline in a concentration of 10 IU/mL until they become engorged. This leads vessels to assume the configuration they will present after being perfused by blood, as when the clamps are removed after anastomoses completion.
Moreover, it is of paramount importance to detect any air bubbles, even if small, inside the vessels during the entire procedure and particularly before tying the final stitches. If these bubbles are distant from the vascular section, the vessels can be milked gently with microsurgical forceps. If they are located close to the anastomotic sites, simple irrigation leads the less dense bubbles to be easily expelled from the vascular lumen. Failure to acknowledge the presence of air bubbles can cause irreversible flap ischemia and necrosis, no doubt due to the fine caliber of the flap vessels. 
Additionally, it cannot be overemphasized the need for meticulous care while passing and tying the stitches, in order to: include the three layers of the vessels (intima, media and adventitia); obtain good vessel eversion to ensure adequate intimal contact, which is vital to anastomosis sealing and endothelial regrowth; avoid loose vascular contact, which will result in anastomotic incompetence, i.e., bleeding; and avoid grabbing too much vascular tissue, which will lead to anastomosis stenosis and proclivity to thrombosis, which in turn will result in venous congestion or poor flap perfusion, if the vein or artery are involved, respectively.
Finally, it is essential to ensure perfect hemostasis, during the entire procedure, especially when raising the flap in its deep surface. Otherwise hematoma formation and rat death are likely to ensue.
Modifications and troubleshooting of the technique
The authors observed that making a transverse incision in the middle portion of the SCM using an electric cautery, not only allows a better exposure of the carotid artery, but also minimizes the risk of undue tension over the future arterial anastomosis.
Another important technical tip is to start the anastomosis from the vessels' back wall, in order to minimize the risk of unwillingly catching this wall when placing the stitches in the more easily exposed front wall. If the back wall is sutured to the anterior aspect of the anastomosis, lack of vascular patency will almost invariably result either immediately due to mechanical reasons or after only a few hours as a result of thrombosis8. 
If the anastomoses of the epigastric vessels of the rat are considered too technically challenging due to the small caliber of these vessels, the femoral vessels can be ligated distal to the origin of the epigastric vessels and used as the vascular pedicle of the epigastric flap. In this way, larger vessels will be used (the femoral artery has a caliber of 1.0 to 1.2 mm; and the femoral vein has a caliber of 1.2 to 1.5 mm). Moreover, by dissecting and ligating the other tributaries of the femoral vessels, a vascular pedicle length of over 2 cm can be obtained, which will facilitate flap insetting18,34,35. 
Reproducibility
Our experience of more than ten years of using this flap for teaching and research purposes strongly suggests that the rat epigastric flap is a reproducible model of free tissue transfer11,13,17,18,26. It can be easily incorporated in microsurgical courses, as it is a good teaching and training model for microsurgery trainees11,13,17,18,26. In our experience, although technically challenging in the beginning for the novice in microsurgery, after some training, the free epigastric flap can be successfully transferred to the neck of the rat with minimal to no subsequent necrosis in 70 to 80% of cases. These results concur with those generally reported in the literature13,18,36. 
Significance with respect to existing methods
Numerous free flaps have been described in the rat10,16,18,37-39. The most commonly used for teaching and research purposes have been the transverse rectus abdominis myocutaneous flap, the latissimus dorsi and serratus anterior muscle flaps, the hind limb replantation model, and the epigastric (groin) flap18,35. These flaps have been favored, due to their consistent anatomy and sizeable vascular pedicle. The epigastric flap is arguably the one associated with lesser donor site morbidity, as it dissected above the muscle fascia18. Moreover, the epigastric flap, described in 1967, was the first flap to be described in rats34,35. This occurred only 4 years after the first description of an experimental flap in an animal by Goldwyn. Interestingly, this flap was a groin flap in the dog34. 
Limitations of the technique
The two main limitations of this model are the need for microsurgical skills in order to carry out the surgery, and the presence of significant necrosis in 20 to 25% of cases, according to different authors13,18,36. Another potential limitation of the model herein presented is the auto cannibalism of the flap. However, as the authors above, this is an infrequent finding that almost only occurs in cases of total flap necrosis.
Future applications of the technique
The rat epigastric free flap can be used in experimental studies of tissue perfusion, tissue repair and surgical wound infection40,41. Its nutrient vessels are particularly suitable for intravascular injection of solutions containing substances of interest, namely drugs, viral vectors or liposomes, that will mostly produce a local or regional effect30,31. In addition, beneath the flap, pathogens, foreign bodies, radioactive seeds or chemicals can also be placed, mimicking several disease processes and potential treatments30,31.

自由組織移動はますます1970 1~5ので欠落組織を再構築するために臨床で使用されてきました。これは、腫瘍摘出、外傷、感染症、奇形や火傷1-7から得られた複合体とそれ以外の治療不可能な欠陥の再構築を可能にしました。この種の無料フラップは頭や首、手、足、および会陰1,4のものと同様、非常に複雑な解剖学的領域を再構築するために特に有用です。 しかし、今日でも外科的研修生は頻繁に顕微技術と機器8,9を使用して自由なフラップを上げ転送しinsettingに関与するいくつかの手順の複雑さによってひるむされています。また、それは広く堪能マイクロ手術の専門医になるためにことが認められている、動物モデルにおいて豊富な実験実習は4,8-13必須です。 また、基本とトランスレーショナルリサーチ無料組織転送の領域に大きな臨床的可能性8,14-16です。それにもかかわらず、研究者が頻繁術4,8-14に関わる技術的側面に関する情報が不足しているため、組織の転送の顕微モデルを使用してから抑止されています。それは比較的、安価で維持しやすく、かつ頻繁な操作8,11,13,14,17,18を受け入れられるように、ラットは、顕微研究と訓練のための優れた動物モデルです。 いくつかの無料の骨、筋肉と皮膚フラップラット18-24に記載されているが、自由な心窩部筋膜皮弁は教育目的9,12,13,18,25のために最も広く使用されています。この無料のフラップはWH、最初Strauchとマレーによって1967年に記述され、いくつかの要因、すなわち一定の血管解剖、解剖の相対的な容易さ、かなりの栄養血管、およびドナーゾーンの皮膚の冗長性に、それ以来増加し人気を得ていますICHは、フラップの標高4,9-11,13,17,18,25-28に起因する欠陥の主要な閉鎖を可能にします。 フラップ解剖学と組織学 上腹部フラップは浅腹壁動脈と静脈（ 図1）によって供給されています。これらの血管は、それぞれに由来し、大腿動脈と静脈に排出します。平均的に浅腹壁静脈の口径は、浅腹壁動脈（ 図2）17,18の0.3〜0.5ミリメートルと対照的な、0.6〜0.8mmです。心窩部の外皮のほとんどを供給毛細血管ネットワークを発信、横方向と順番に複数回分割内側枝：浅腹壁動脈は、2つの主要な枝を放ちます。これらの毛細血管は、動脈樹（ 図2）13,17,18と平行のコースを持っている浅腹壁静脈の支流に排出します。 図3の再のダイアグラム上腹部のフラップに動員することができ浅上腹部管により供給された腹腹壁の領域を示しています。このフラップは、長さ5cm、幅13,17,18の3センチまで可能です。 組織学的には、フラップは腹外側腹壁の筋肉（ 図4）13,17,18 をカバー外皮で構成されています。これは、真皮と表皮によって形成された皮膚の表面層を、含まれています。皮膚の下に脂肪の皮筋層という名前の脂肪組織の層が存在します。この層の下に皮筋層 18,28,29として知られる横紋筋の別の層が存在します。 皮筋層の下には、より大きな腹部の筋肉をカバーし、深い筋膜に表面的であるゆるい疎性結合組織があります。したがって、フラップは（ 図5）1深部筋膜を除いて、すべてのこれらの層を含む、組織の複合ブロックであります3,17,18,27-31。

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			<td>http://www.expresschemist.co.uk/lacri-lube-eye-ointment-5g.html</td>
		</tr>
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			<td>Normal saline for irrigation</td>
			<td>Hospira, Inc.</td>
			<td>0409-6138-22</td>
			<td>http://www.hospira.com/en/search?q=sodium+chloride+irrigation%2C+usp&#38;fq=contentType%3AProducts</td>
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			<td>Heparin Sodium Solution (5000IU/ml)</td>
			<td>B.Braun</td>
			<td></td>
			<td>http://www.bbraunusa.com/products.html?prid=PRID00006982</td>
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			<td>Meloxicam Metacam</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>http://www.bi-vetmedica.com/species/pet/products.html</td>
		</tr>
		<tr>
			<td>Heat Lamp HL-1</td>
			<td>Harvard Apparatus</td>
			<td>727562</td>
			<td>https://www.harvardapparatus.com/webapp/wcs/stores/servlet/ 
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			37610_37611_37613</td>
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			<td>Homeothermic Blanket System with Flexible Probe</td>
			<td>Harvard Apparatus</td>
			<td>507220F</td>
			<td>https://www.harvardapparatus.com/webapp/wcs/stores/servlet/ 
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			37610_37611_37613</td>
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			<td>Dry heat sterilizer</td>
			<td>Quirumed</td>
			<td>2432</td>
			<td>http://www.quirumed.com/pt/material-de-esterilizac-o/esterilizadores</td>
		</tr>
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			<td>Surgical drapes</td>
			<td>Barrier</td>
			<td>800430</td>
			<td>http://www.molnlycke.com/surgical-drapes/</td>
		</tr>
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			<td>Biogel Surgical Gloves</td>
			<td>Medex Supply</td>
			<td>30465</td>
			<td>https://www.medexsupply.com</td>
		</tr>
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			<td>Operating microscope</td>
			<td>Leica Surgical Microsystems</td>
			<td>10445319</td>
			<td>http://www.leica-microsystems.com/products/surgical-microscopes/</td>
		</tr>
	</tbody>
</table>

a model of free tissue transfer: the rat epigastric free flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

顕微コースのコンテキスト内や研究目的のために、両方の無料組織転送のモデルとして上腹部無料フラップを使用して、十年以上の著者の経験によると、フラップの生存率は、外科医の器用さと経験にやや依存します。上述の技術的側面を考慮した場合、一般的に言えば、ほぼ完全な生存率（&lt;フラップ壊死の10％）フラップの約70％が予想されます。フラップの約10％は、部分的な壊死（10〜50％）を提示します。フラップの約20％は完全壊死に苦しみます。 80％ほぼ完全な生存率は、第一著者（DC）（ 図13）によって実行された最後の20の手順で得られました。 術後最初の2日間は、無料の上腹部フラップは、多くの場合、浮腫状であり、静脈うっ血ある程度のを提示します。これらの通常ボット時間は手術後3〜5日の間に徐々におさまります。一般的に、最初の週の間、ラットは、多くの場合、わずかな創傷裂開の散乱領域（ 図14）、その結果、ほとんどの外部縫い目や表皮下縫合糸の一部を削除します。 10日目の後、髪はゆっくりとフラップの表面に成長を開始します。手術後の最初の月の終わりには、フラップは通常、隣接する皮膚よりもわずかに短い毛で覆われています。二ヶ月は術後、フラップの存在はわずかな塊により、フラップの縁の周りに比較的目立たない傷跡（ 図14）によって予告されています。フラップの自動共食いは、著者の経験では、総フラップ壊死の例でほぼ独占的に発生し、まれな知見です。 <img alt="図1" src="/files/ftp_upload/55281/55281fig1.jpg" /> 図1： 心窩部の無料FLAの血管解剖学P。 この写真は、以前に動脈系に赤いラテックス溶液とし、静脈系における青色ラテックス溶液を注射したラットの左心窩部を示しています。上腹部領域が浅腹壁動脈と静脈から軸血液供給を受けることを観察することができます。これらの血管は、それぞれに由来し、大腿動脈と静脈に排出します。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig1large.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2" src="/files/ftp_upload/55281/55281fig2.jpg" /> 図2： 上腹部無料フラップに微細な血管の血液供給を示す浅上腹部管の腐食キャストの走査型電子顕微鏡像。 superfiの腐食キャストのこの走査型電子顕微鏡像ラットの公式な上腹部血管は静脈は動脈より大きな口径を有していることを示しています。平均的に浅腹壁静脈の口径は、浅腹壁動脈の0.3〜0.5ミリメートルと比較して0.6〜0.8mmで、です。心窩部のほとんどを供給する毛細血管のネットワークを発信、横方向と順番に複数回分割内側枝：この画像はまた、浅腹壁動脈は二つの主枝を発信することを示しています。これらの毛細血管は、動脈樹に平行コースを持っている浅腹壁静脈の支流に排出します。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig2large.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/55281/55281fig3.jpg" /> 図3：ラットにおける左上腹部無料フラップの潜在的なエリア。 この図のrepre浅上腹部管により供給された腹壁の領域にsents、それは上腹部フラップに動員することができます。このフラップは、長さ5cm、幅3センチまで可能です。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig3large.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4" src="/files/ftp_upload/55281/55281fig4.jpg" /> 図4： 上腹部フラップのヘマトキシリン-エオシン染色切片の写真。 心窩部のこのヘマトキシリン - エオシン染色したセクションでは、上腹部フラップは腹壁の筋肉をカバーし、この領域の外皮で構成されていることを示しています。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig4large.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> 図5： 上腹部フラップの組織学的組成物。 右側の写真は、このフラップのマッソン三色のセクションから入手したのに対し、左側の写真は、上腹部フラップのヘマトキシリン - エオシン染色したセクションを表します。これら二つの絵は、ラットの上腹部フラップは組織の複合ブロックであることを示しています。これは、真皮と表皮によって形成された皮膚の表面層を、含まれています。皮膚の下に脂肪の皮筋層という名前の脂肪組織の層が存在します。この層の下に皮筋層として知られる横紋筋の層が存在します。 皮筋層の下には大きくて深い腹部の筋肉をカバーし、深い筋膜があります。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig5large.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6" src="/files/ftp_upload/55281/55281fig6.jpg" /> 手術前に 、図 ラットの腹面6.術前皮膚マーキング。 この写真は、左上腹部フラップを上げるために、続いて左頸部の腹側面では、このフラップをはめ込むために使用される切開のための皮膚マーキングを示しています。 <img alt="図7" src="/files/ftp_upload/55281/55281fig7.jpg" /> 図7. 手術用顕微鏡（倍率10倍）の下で上腹部フラップの栄養血管の外科解剖学。 この写真は、浅腹壁動脈と静脈から発信され、それぞれ、大腿動脈と静脈に排水示しています。外側大腿回旋動脈は通常、表面的​​なエピの尾側の側面から生じます胃動脈。外側大腿回旋静脈は、同様の経路を有しており、通常は浅腹壁静脈内に終了します。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig7large.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8" src="/files/ftp_upload/55281/55281fig8.jpg" /> 図8. その栄養血管に有茎 上腹部フラップ ex vivoで （浅腹壁動脈と静脈 - A、V、それぞれ）。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig8large.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9" src="/files/ftp_upload/55281/55281fig9.jpg" /> の図9.オペレーティング・ビュー受信者の静脈の解剖、すなわち、首の左側の外頚静脈、（6倍の倍率）。 胸鎖乳突筋、外部頸静脈の側面の皮下の経過を観察することが可能です。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig9large.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10" src="/files/ftp_upload/55281/55281fig10.jpg" /> 首（10倍の倍率）の左側のドナー動脈、すなわち、総頸動脈の解剖図 10. オペレーティング・ビュー、。 示すように、動脈および添付迷走神経は、胸鎖乳突筋と舌骨下筋を後退させた後に公開されています。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig10large.jpg" target="_blank">トン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>彼の姿。 <img alt="図11" src="/files/ftp_upload/55281/55281fig11.jpg" /> 手術用顕微鏡（10倍の倍率）の下で見られるように、フラップの血管や首の受信者の容器間の血管吻合、 図 11. 写真。 この写真は、総頸動脈と浅腹壁動脈との間termino横吻合を示しています。表面的な心窩部と外頸静脈の間termino末端吻合を観察することも可能です。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig11large.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12" src="/files/ftp_upload/55281/55281fig12.jpg" /> ラットimmediの腹側面の 図12. 写真<html> ately手術後。 ドナーゾーンを簡単に主に閉鎖されることに注意してください。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig12large.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13" src="/files/ftp_upload/55281/55281fig13.jpg" /> 20連続したラットにおける 図13. 心窩部無料弁の生存は、第一著者（DC）によって操作しました。 5匹のラット（20％）は、完全なフラップ壊死（赤い点で示される場合に1、4、8、13及び15）に提示しました。トルヒョらによって詳細に説明するように、フラップ壊死の領域は、フリーソフトウェアのImageJを用いて決定しました。 15。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig13large.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> 14 &quot;SRC =&quot; /ファイル/ ftp_upload / 55281 / 55281fig14.jpg &quot;/&gt; 術後 図14 首4の腹側面に配置された上腹部フラップの写真、14、60日。 ラットがステッチを削除するように4日手術後、典型的には、いくつかの創傷裂開があります。しかし、フラップは通常、所定の位置に残ります。単純な目視検査により毎日フラップを調べることができます。 <a href="http://ecsource.jove.com/files/ftp_upload/55281/55281fig14large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about A Model of Free Tissue Transfer: The Rat Epigastric Free Flap at JoVE.com

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

無料組織転送のモデル：ラット心窩部無料フラップ

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

a-model-of-free-tissue-transfer-the-rat-epigastric-free-flap

Research

JoVE Journal

Medicine

16.6K ビュー.  Universidade NOVA de Lisboa. This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

ビデオ: A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

α-テスト：全唾液を用いた迅速な無細胞CD4列挙

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

ラットにおける慢性栄養注入のモデル

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

筋皮虚血再灌流傷害のラットモデル： 現場横腹直筋皮フラップで

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

非接触型誘電泳動によりラベルフリーの単離と細胞の濃縮

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bell&#8217;s palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis.
The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity.
Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

The use of the masseteric nerve as donor nerve represents a single-stage alternative to the criterion standard two-stage procedure of cross-facial nerve grafting and free muscle transfer in facial paralysis. We provide a detailed description to safely perform this technique with a gracilis muscle transfer and discuss indications and limitations.

フリー機能性筋肉転送により不可逆顔面神経麻痺が笑うの単段ダイナミック蘇生

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

ウェルシュ病原性を研究する麻酔特定病原体フリー鶏における腸管結紮ループ モデル

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

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 ...

無料組織転送のモデル：ラット心窩部無料フラップ

要約

さらに動画を探す

この動画の章

α-テスト：全唾液を用いた迅速な無細胞CD4列挙

CD133の患者由来の細胞培養および分離

ラットにおける慢性栄養注入のモデル

筋皮虚血再灌流傷害のラットモデル：現場横腹直筋皮フラップで

非接触型誘電泳動によりラベルフリーの単離と細胞の濃縮

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

フリー機能性筋肉転送により不可逆顔面神経麻痺が笑うの単段ダイナミック蘇生

共焦点光学イメージングによるローズベンガル光血栓

ウェルシュ病原性を研究する麻酔特定病原体フリー鶏における腸管結紮ループモデル

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

無料組織転送のモデル：ラット心窩部無料フラップ

要約

さらに動画を探す

この動画の章

α-テスト：全唾液を用いた迅速な無細胞CD4列挙

CD133の患者由来の細胞培養および分離

ラットにおける慢性栄養注入のモデル

筋皮虚血再灌流傷害のラットモデル： 現場横腹直筋皮フラップで

非接触型誘電泳動によりラベルフリーの単離と細胞の濃縮

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

フリー機能性筋肉転送により不可逆顔面神経麻痺が笑うの単段ダイナミック蘇生

共焦点光学イメージングによるローズベンガル光血栓

ウェルシュ病原性を研究する麻酔特定病原体フリー鶏における腸管結紮ループ モデル

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

筋皮虚血再灌流傷害のラットモデル：現場横腹直筋皮フラップで

ウェルシュ病原性を研究する麻酔特定病原体フリー鶏における腸管結紮ループモデル