著者は、国民健康とオーストラリアの医学研究評議会（NHMRC）（：632512プロジェクト無償ID）から資金援助を承認したいと思います。ルイーズダン、国立心臓財団キャリア開発フェローシップによってNHMRC早いキャリアフェローシップとクリスティーナBursillによってサポートされていました。

1。スプリントと閉塞性ドレッシングの調製<ol><li>シリコンディスクを作成するために厚さ0.5mmのシリコンシートと利用ハサミや生検パンチで10ミリメートルの円を概説。 </li><li>センター10ミリメートルサークルの真ん中に5ミリメートル生検パンチと添え木として使用され、 &quot;ドーナツ&quot;のような円盤を形成するための穴を作成するためにしっかりと押します。 </li><li>などOpsiteなどの透明密封包帯に10ミリメートルの円を概説し、円形のドレッシングを作成するためにハサミを使用しています。 </li></ol> 2。実験動物<ol><li>実行されるすべての実験のための動物倫理委員会の承認を得る。 </li><li>商業ブリーダー（ 例えばジャクソン研究所）から8週齢（22〜26グラム）の雄C57BL/6Jマウスを使用してください。 </li><li> 21の標準的な条件でマウスを保つ°Cと食物と水に自由にアクセスして12時間の明暗サイクル。 </li><li> （オプション）：糖尿病はボーラス腹腔麟蹄で6から7週齢のマウスに誘導することができる定期的な血糖測定（AccuChekグルコメーター）により確認高血糖と165 mg / kgでストレプトゾトシン（クエン酸緩衝液中で、pH4.5）中のction。糖尿病マウスでは、尿があるかもしれませんので、それらの寝具は湿気を排除するために、より頻繁に変更する必要があるかもしれませんし、その重みが密接に監視する必要があります。 </li></ol> 3。麻酔と手術の準備<ol><li> 100％酸素でイソフルラン5％（流量1リットル/分）を使用して、全身麻酔を誘導し、イソフルラン1〜3％を使用して麻酔を維持します。 </li><li>マウスの深いペダル反射が抑制されていることを確認し、発生しやすい位置にマウスを置きます。 </li><li>首の付け根から背中ダウンと2つの肩甲骨の間のさらに3 cmまでバリカンで毛を除去することにより、手術部位を準備します。 </li><li>脱毛クリームの光照射は、2分を超えないために適用することができる。ウェットガーゼ綿棒は、すべてのクリームを確保するために使用することができ、残りの毛を除去する。 </li><li>で皮膚を拭きアルコール綿棒、10％ポビドンヨードの二つのアプリケーション（ベタジン）とマウスをドレープ。 </li></ol> 4。傷の切除と副木<ol><li>マウスの創傷治癒モデルの概略図を図1に示されている。 </li><li>肩のレベルで、マウスの正中線の両側に傷のために2つの円形のパターンを概説する無菌4ミリメートル生検パンチを使用してください（ 図2a-2b参照 ）。 </li><li>皮筋層（ 図2E）を含む皮下組織（ 図2C-2D）を通って延びて全層傷、および消費税円形の作品を作成するためにアウトラインとアイリスのはさみの真ん中に皮膚を持ち上げるために鋸歯状の鉗子を使用して、組織の。 </li><li>正中線（ 図2F）の反対側の傷のためにプロセスを繰り返します。 </li><li>シリコーンスプリントの各側面からプラスチックの保護コーティングを除去。 </li><li> cyanoacrylaを適用teの接着剤（ 例えば、スーパー接着剤またはクレイジー接着剤）シリコーンスプリントの片側に。 </li><li>センターでは、傷の上に添え木（ 図2G）と位置決め（ 図2H）を確実にするために中断された6-0ナイロン縫合糸で添え木を固定。 </li><li>他の傷に副木プロセスを繰り返します。 </li><li>この時間の時点で必要に応じて、もう一方の傷（ 図2I）と、車両制御に（最大100μl）をテストする治療化合物を適用します。 </li><li>透明密封包帯（例えば、OpSite）（ 図2J-2K）で傷口をカバーしています。 </li><li>定規は、スプリントと（2リットル図 ）とら顕微鏡写真の下方に配置されている。 </li></ol> 6。術後管理<ol><li>カルプロフェンは（5 mg / kg）を術後の疼痛緩和のための皮下注射により1日1回投与する。 </li><li>手術動物は個別にケージと熱メートル上に維持された後ATSは完全に回復するまで。 </li><li>痛みや体重減少の症状のために二回、毎日動物を監視します。我々は、痛みや体重減少の肉眼行動ディスプレイは観察されなかった。 </li></ol> 7。傷の測定と治療<ol><li>傷は、毎日測定することができます。 </li><li> 5％イソフルランガス（流量1リットル/分）を使用して、全身麻酔を誘導した後、マウスの深い感覚の反射を確実に1〜3％がイソフルラン使用して抑制されます。 </li><li>ピンセットでそっと裏閉鎖包帯。 </li><li>巻径を測定するために外科キャリパーを使用してください。私たちは、X、YおよびZ軸方向（ 図2メートル-20）と同様に将来の参照（ 図2P）のための顕微鏡写真に沿って3つの測定値の平均を取る。 </li><li>オプション：この時点で動物はレーザードップラーイメージャ（ 図2Q）を使用して、血流のために評価することができる。 </li><li>治療用化合物と車両制御の再アプリケーションがパフォーマンスすることができますこの時点でMED。 </li><li>きれいな透明閉鎖包帯は、その後再適用し、完全に回復するまでの動物は保温されています。 </li><li>注：スプリントが適切に確保されていない場合、傷は容易に収縮する（ 図2R）。 </li></ol> 8。組織学的分析<ol><li>麻酔の過剰摂取で10日後のマウスを安楽死させる。 </li><li>鉗子やメスの刃を使用すると、縫合糸、慎重剥がれ添え木を削除します。 </li><li> 4℃で一晩緩衝生理食塩水（PBS）創傷領域の周りや下に広い、完全切除を作成し、リンの4％パラホルムアルデヒドで組織をインキュベートするアイリスのはさみを使用しています。 </li><li> °C、その後-80℃でOCTコンパウンド、凍結、組織や場所に慎重に軽くたたくことで過剰の溶液を除去し4℃でさらに24時間PBS溶液中で17％ショ糖に組織を移す</li><li>ヘマトキシリン​​およびエオシン染色、創傷構造および上皮ギャップを可視化するために使用することができる。 Neovascularisationは、毛細管の数（フォンビルブラント因子を使用して）決定するために免疫組織化学的分析によって評価することができる。 </li></ol>

<ol>
	<li>Sen, C. K., 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=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair. 17, 763-771 (2009).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17, 1-18 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Lindblad, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+selecting+the+correct+animal+model+for+dermal+wound-healing+studies.">Considerations for selecting the correct animal model for dermal wound-healing studies.</a> J. Biomater. Sci. Polym. Ed. 19, 1087-1096 (2008).</li><li>Grose, R., Werner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+studies+in+transgenic+and+knockout+mice.">Wound-healing studies in transgenic and knockout mice.</a> Mol. Biotechnol. 28, 147-166 (2004).</li><li>Reid, R. R., Said, H. K., Mogford, J. E., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+wound+healing:+pursuing+surgical+models+in+transgenic+and+knockout+mice.">The future of wound healing: pursuing surgical models in transgenic and knockout mice.</a> J. Am. Coll. Surg. 199, 578-585 (2004).</li><li>Fang, R. C., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+healing:+utility+in+transgenic+mice.">Animal models of wound healing: utility in transgenic mice.</a> J. Biomater. Sci. Polym. Ed. 19, 989-1005 (2008).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> J. Biomed. Biotechnol. 2011, 969618 (2011).</li><li>Galiano, R. D., Michaels, J. t., Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair. 12, 485-492 (2004).</li><li>Galiano, R. D., 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=Topical+vascular+endothelial+growth+factor+accelerates+diabetic+wound+healing+through+increased+angiogenesis+and+by+mobilizing+and+recruiting+bone+marrow-derived+cells.">Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.</a> The American Journal of Pathology. 164, 1935-1947 (2004).</li><li>Thangarajah, H., 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=The+molecular+basis+for+impaired+hypoxia-induced+VEGF+expression+in+diabetic+tissues.">The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13505-13510 (2009).</li><li>Raza, A., Bayles, C., Biebel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Bacterial+Growth+and+Moisture+Handling+Properties+of+Transparent+Dressings:+3M+Tegaderm+Transparent+Dressing,+3M+Tegaderm+HP+Transparent+Dressing,+and+Opsite+IV3000+Transparent+Dressing.">Investigation of Bacterial Growth and Moisture Handling Properties of Transparent Dressings: 3M Tegaderm Transparent Dressing, 3M Tegaderm HP Transparent Dressing, and Opsite IV3000 Transparent Dressing.</a> Smith and Nephew Report. , (1998).</li><li>Chung, T. Y., Peplow, P. V., Baxter, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+photobiomodulatory+effects+of+laser+irradiation+on+wound+healing:+development+of+an+improved+model+for+dressing+wounds+in+mice.">Testing photobiomodulatory effects of laser irradiation on wound healing: development of an improved model for dressing wounds in mice.</a> Photomed. Laser Surg. 28, 589-596 (2010).</li></ol>

著者らは、開示することは何もありません。

これは、皮膚の創傷治癒の実験マウスモデルである。このモデルの重要な特徴は、ヒトで発生するプロセスと、それが類似すること、再上皮化し、新しい組織の形成が起こることができるように、創傷収縮を防ぐために、シリコーン副木の使用である。このモデルは、用途が広く、生理学的および病態生理学的の両方（ 例えば、糖尿病）の設定における創傷治癒を評価するために使用...

障害創傷治癒にはかなりの罹患率と死亡率を世界的に責任があるが、これは糖尿病1,2の患者のために特に当てはまります。ヒトでは、創傷治癒は3と重なる重要な存在である、工程の連続である。直後に負傷、炎症プロセスが開始されます。炎症細胞は、細胞増殖、遊走及び血管新生の過程を促進する因子を放出する。再上皮化し、新しい組織の形成後にコラーゲンなどのマトリックスタンパク質のアポトーシス及び再組織の両方を伴うリモデリングの段階がある。 創傷治癒の複雑性は、現在、 インビトロで複製され、これは、動物モデルの使用を必要とすることはできない。現在までに、創傷治癒研究は治癒過程が同等と人間に匹敵することを保証するために、例えば、ブタなどの大型動物モデルが、これらに限定されている。しかし、大規模なアニマを使用このような研究のためのlsが家に困難になり、常に実用的4ではありませんすることができます。実験用マウスは簡単に遺伝的に5-7序調査のために操作することができる経済的な動物モデルを表しています。しかし、ネズミの傷は人間の、収縮8の過程が主な原因とは異なって癒す。これは、部分的にヒトに主として存在しないか、皮筋層と呼ばれる広範な皮下横紋筋層によるものである。マウスでは、この筋層は、皮膚が独立深い筋肉の移動することができ、皮膚の次の負傷の急速な収縮を担当しています。 この制限を克服するために、マウスの創傷治癒は、スプリント（ 図1）8,9を用いてヒトの創傷治癒を複製するように構成することができる。このビデオでは、創傷収縮がなくなり、より密接に再epithelializa人間の過程を近似splintedマウス創傷モデルを実証ると新しい組織形成。このモデルでは、皮筋層を含む2つのフル厚切除がマウスの正中線の両側に背、1で作成されます。シリコーンスプリントは、接着の支援、その後中断した縫合糸で固定スプリントで傷の周囲に配置されています。各マウスことにより、動物の数を減少させる、傷一つは、治療およびその他の車両制御を受け取ると、自身のコントロールとして作用する。局所適用に続いて、透明密封包帯を適用する。さらに、局所適用および/ ​​または創傷領域10,11の測定に必要なときに包帯を除去することができる。実験では、創傷閉鎖の終了時に、血管新生の形態アーキテクチャおよび程度は、免疫組織化学により評価することができる。この経済的モデルを実行することが容易にも糖尿病または他の病態生理との関連で創傷治癒を評価するために利用することができる。

<table border="0">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Press-to-seal silicone sheeting 0.5 mm thick</td>
 <td>Invitrogen</td>
 <td>P18178</td>
 <td>Cut into "donuts" with external diameter of 1cm external, 0.5 cm internal diameter</td>
 </tr>
 <tr>
 <td>Biopsy punch 5 mm</td>
 <td>Steifel</td>
 <td>BC-B1-0500</td>
 <td>To outline wound area to be excised</td>
 </tr>
 <tr>
 <td>Vannas scissors 8.5 cm curved</td>
 <td>World Precision Instruments </td>
 <td>501232</td>
 <td>For wound incision and excision</td>
 </tr>
 <tr>
 <td>Dumonte #7b forceps, 11 cm</td>
 <td>World Precision Instruments</td>
 <td>501302</td>
 <td>To grip skin when creating incision and excising skin </td>
 </tr>
 <tr>
 <td>Graefe forceps, serrated 10cm</td>
 <td>World Precision Instruments</td>
 <td>14142</td>
 <td>To help attach silicone splint to skin</td>
 </tr>
 <tr>
 <td>Needle holder, smooth jaws, curved, 12.5 cm</td>
 <td>World Precision Instruments</td>
 <td>14132</td>
 <td></td>
 </tr>
 <tr>
 <td>Malis forceps, smooth, straight, 12 cm</td>
 <td>Codman and Shurtleff, Inc (J&amp;J)</td>
 <td>80-1500</td>
 <td>To suture the silicon rings to the skin</td>
 </tr>
 <tr>
 <td>Ruler, 0.5 mm gradation</td>
 <td>n/a</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calipers 0.25 mm gradation</td>
 <td>Duckworth and Kent</td>
 <td>9-653</td>
 <td>To measure wound area</td>
 </tr>
 <tr>
 <td>Opsite FlexiFix transparent adhesive film. 10 cm x 1 m</td>
 <td>Smith &amp; Nephew</td>
 <td>66030570</td>
 <td></td>
 </tr>
 <tr>
 <td>Rimadyl (Carprofen)</td>
 <td>Pfizer</td>
 <td>462986</td>
 <td></td>
 </tr>
 </tbody>
</table>

murine model of wound healing

創傷治癒と修復は、人間の生活の中で発生する最も複雑な生物学的プロセスである。怪我の後、複数の生物学的経路が活性化。例えば糖尿病患者に起こる障害創傷治癒は、このような切断などの重篤な不利な結果につながることができます。創傷修復を促進する新規な薬剤を開発するために増加させる原動力は、したがって、がある。これらのテストは、例えば、しばしば非実用的であるブタなどの大型動物モデルに限られていた。彼らは経済的で機械的な調査が可能に遺伝子操作に適しているようにしたマウスは、理想的な前臨床モデルを表しています。しかし、マウスにおける創傷治癒は、それが主に収縮を介して行われるように、人間のそれとは根本的に異なる。我々のマウスモデルは、創傷の周りに添え木を組み込むことによって、これを克服する。傷を副木で、修復プロセスが密接にミラー上皮化、細胞増殖や血管新生に次に依存しているヒトの創傷治癒の生物学的プロセス。一貫性とケアを必要とする一方で、このマウスモデルは、複雑な手術手技を伴い、例えば、血管新生を促進したり、炎症を阻害する可能性がある有望な薬剤の堅牢なテストのためにできません。 2創傷を調製するようさらに、各マウスは、試験化合物と同じ動物上の車両制御の両方のアプリケーションを可能にし、それ自体の対照として作用する。結論として、私たちは、人間に匹敵する創傷治癒の実用的に学ぶことが簡単に、かつ堅牢なモデルを示しています。

（0日目径/日のX径） -創傷閉鎖曲線は、創傷の平均直径を算出し、比率、 すなわち 100として結果を表現することにより決定される。この実験では、治療化合物（またはビヒクル対照）創傷に毎日適用した。治療化合大幅に加速された創傷閉鎖（ 図3）。これはスプリントの除去が迅速創傷収縮（ 図2R）につながり、ヒトで観察された創傷治癒のパターンから?...

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Murine Model of Wound Healing

生理学的および病態生理学的環境における治療用化合物を評価するために使用され得る皮膚の創傷治癒のマウスモデル。

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. ...

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63.5K ビュー.  The Heart Research Institute. 生理学的および病態生理学的環境における治療用化合物を評価するために使用され得る皮膚の創傷治癒のマウスモデル。

ビデオ: Murine Model of Wound Healing

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

マウス内視鏡検査のため<em&gt;インビボ</em&gt;発がんと腸の評価のマルチモーダルイメージング創傷治癒および炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

医学

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

ラット虚血皮膚創傷モデルの実証

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

デジタルホログラフィック顕微鏡とマルチモーダル定量位相イメージングは​​、正確に腸の炎症および上皮創傷治癒を評価します

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

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 ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

顕微鏡法によるIn Vitro創傷が治癒過程における上皮移行の評価

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

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 ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.