Name: come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model
Uploaded: 2017-10-10T13:00:00.000+00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

著者らはこの作業 (グラント # #7-13-BS-180)、トレーニングと、生体内でイメージ投射システムへのアクセスを提供するためミシガン州立大学研究技術支援施設を支援する米国糖尿病協会を感謝したいとミシガン州大学調査病理組織学的研究室マウス生検病理組織学的検査のためを処理するため

動物実験は機関動物ケアおよび使用委員会のミシガン州立大学によって承認されました。
 1. 閉塞性ドレッシングの準備やシリコーン スペーサー <ol><li>透明な閉塞性ドレッシングをカットして正方形約 1 cm × 1 cm ハサミで</li>。
	<li>カット 10 mm 丸 10 mm 生検パンチ センター 5 mm 生検を使用して 0.5 mm 厚のシリコン シート 10 mm の円の中にパンチとしっかりとフォームに穴を作成するを押して、&#34; ドーナツ &#34;-添え木として使用されるディスクのよう。</li></ol>。
 2. 実験動物 商業ブリーダーから <ol><li>使用 8 週間の古い (22-26 g) 男性 SKH 1 マウス。食料と水 21 ° C と無料で 12 時間の明暗周期の標準条件でマウスを維持します</li>。
	<li>糖尿病を誘発する注入マウス intraperitonially 13 mg/ml ストレプトゾトシン (STZ) solutionand 25% ブドウ糖 (250 μ l/マウス/) 5 日間連続で。
	<ol><li>100 mm クエン酸、pH 4.5 5 μ L の STZ の希釈の 65 μ g で STZ ソリューションを作成します</li>。
		<li>は、マウスの体重の 1 kg あたり 65 mg STZ の最終的な質量に各マウスの噴射量を調整します。同じ日に 100 mM クエン酸溶液 pH 4.5 でマウスを挿入します</li>。
	</ol></li> <li>血糖モニタリング、glucometer と最後の STZ 投与後 14 日間で高血糖を確認します。多尿があります糖尿病マウスそして彼らの寝具は湿気を除去するためにはより頻繁に変更する必要があります、その重みを週に 3 回を監視します</li>。
</ol> 3。バイオ フィルム <ol><li>バイオ フィルム準備 <ol><li>成長のコロニー バイオ フィルム 8 傷を接種します。37 ° C で培養した手術開始前に 2 日間発光の一晩文化 膿 Xen トリプシン大豆スープ (TSB) 41 200 rpm で揺れと紫外線への露出によって 0.2 μ m 孔サイズのポリカーボネート製メンブラン フィルターを殺菌しなさい側あたり 15 分のための生物学的安全フードの光</li>。
		<li>1 日手術前に遠心 2 分 20,000 × g で一晩かけて培養し、Dulbelcco 1 mL で 3 回洗う &#39; 上下ピペッティングによる s リン酸緩衝生理食塩水 (DPBS).</li>
		<li>DPBS 600 0.05 の吸光度に懸濁液を希釈 nm</li>。
		<li>トリプシン大豆寒天 (TSA) に各膜希薄文化のピペット 10 μ L。乾燥することができます後、72 時間ごと 24 時間新鮮な TSA プレートに転送するバイオ フィルムの成長を 35 ° C で膜を孵化させなさい</li>
	</ol></li> <li>標準曲線 <ol><li>発光と細菌数を関連付ける標準曲線を作る実験の開始前にします</li>。
		<li>3.1 で述べたように、バイオ フィルムを準備します</li>。
		<li>は、視覚的に均一溶液が作成されるまで DPBS とボルテックス バイオ フィルムを混合することにより、1/2 から 1/24 に至るまでバイオ フィルムのシリアル希薄を作る。黒の 96 ウェル プレートと 生体内 イメージング システム イメージで希薄水溶液のピペット 200µL.</li>
		<li>TSA のプレートにプレートの希釈液を広げ、35 ° C 24 時間インキュベート</li>
		<li>プレートのコロニー形成単位 (CFU) をカウントし、生物発光と細菌数を関連付ける標準曲線を作成します</li>。
	</ol></li></ol> 4。手術の傷 <ol><li>1 L/分の流量で 95% oxygen/5% CO 2 (性ケトアシドーシスによる死亡を防ぐため) にイソフルランを使用して全身麻酔を誘導し、1-3% イソフルランを使用して麻酔を維持します。手術中に熱マット上の動物を維持します</li>。
	<li>マウスの深いペダル反射足をピンセットでつまんで抑制されやすい位置にマウスを置きことを確認します</li>。
	<li>メロキシカム (0.2 mg/kg) を皮下注射 (30 μ l) 痛みの管理を介して管理します</li>。
	<li>10% ポビドン ヨード 3 回で背中とイソプロパノール パッドの肌を拭く</li>。
	<li>マウスの 1 つの側面の傷の 1 つの円形のパターンの概要を説明する滅菌 4 mm 生検パンチを使用して &#39;、肩のレベルで s 正中線。永久的なマーカーとパターンの概要を説明します</li>。
	<li>鋸歯鉗子を使用して組織層 carnosus を含む皮下組織まで全層創傷を作成し、組織の円形の部分を切除する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ます.</li>
	<li>は、医療防水皮膚接着剤接着剤をマウスの皮膚に適用し、穏やかな圧力を適用するシリコーン スプリントを配置します。透明な閉塞性ドレッシング材で傷をカバーしてください。手術後、個別にケージ動物</li>。
</ol> 5。術後管理 <ol><li>メロキシカム (0.2 mg/kg) 一度毎日皮下注射を介して次の 2 日間の術後鎮痛管理します</li>。
	<li>モニター動物の痛み、体重減少の症状のために毎日。糖尿病動物体重の 15% 以上を紛失した場合インスリン注射が必要です</li>。
</ol> 6。バイオ フィルムの接種材料の準備や感染症 <ol><li>次の手順で説明するよう手術後 48 時間のマウスに接種します</li>。
	滅菌のへらを使用して膜から <li>こすり 72 h バイオ フィルム微量遠心チューブに配置し、希釈 DPBS で 1:2。簡単に上下にピペッティングしてミックスします</li>。
	<li>拡散板合計 CFU を計算する TSA のプレートに接種。カウントが正確であるためには、一連の超音波洗浄器で 40 kHz で 2 分間超音波処理ステップによってインターカ レートした 2 つ 1 分ボルテックス手順によってさらにバイオ フィルム菌を打破します</li>。
	<li>ドレッシングでカバー傷とシリコーン スプリントし、参照の定規を使用して接続されているカメラで顕微鏡と傷口の顕微鏡写真を取るを削除します</li>。
	<li>200 μ L ピペット チップの先端をカットし、各傷にバイオ フィルム菌の 10 μ L をピペットします</li>。
	<li>生体内 イメージング システムの自動設定を使用してイメージのマウス: 露光時間 5-300 s、中ビン、1 f/ストップと開いているフィルター ビューのフィールド C と (12.9 x 12.9 cm).</li>
	<li>カバーは新鮮なドレッシング創傷します</li>。
</ol> 7。傷の計測とイメージング <ol><li>動物毎日 9 の臨床徴候を評価します</li>。
	<li>食品を提供、水、ケージを必要に応じて変更します</li>。
	毎日のドレッシングの <li>チェックの整合性。ドレッシングが存在する場合は、傷の閉塞のためのみ発光を測定できます。8 日でドレッシングが削除され、傷の閉鎖のできる測定を置き換えられません</li>。
	<li>その他の毎日の動物の重量を量ます</li>。
	<li>、すべての日の隔離室で個別にマウスを配置HEPA フィルターと、生体内で イメージ投射システム毎日または発光値がバック グラウンド レベルを下回るまで、その他の毎日を使用してイメージを装備</li>。
	<li>8 日後全身麻酔を誘導し、傷が治癒するまで毎日参照の定規を使用して接続されているカメラで顕微鏡と傷口の顕微鏡写真を撮ます</li>。
</ol> 8。組織学的解析 <ol><li>安楽死、安楽死の CO 2 の 2 l/分の流量でマウスのバック グラウンド レベルを下回ると発光、傷が完全に治癒後の部屋。2 番目のメソッドとして安楽死の頚部転位によって死を確認します</li>。
	<li>アイリスはさみを使用しての周りや創傷部 (直径約 1 cm) 下の広い、完全切除を作成し、組織学的解析のための 4% パラホルムアルデヒドで組織を維持します</li>。
	<li>他の分析: サイトカインの検出 <ol><li>レトロ軌道毛細管ガラス管を用いた麻酔下の動物から血を収集し、EDTA 処理管にそれを転送します</li>。
		<li>4 ° C で 20 分間 2,000 rpm で血液を遠心し、サイトカインの検出のため血漿を凍結します</li>。
	</ol>
	</li>
</ol>

<ol>
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著者は、彼らは競合する金銭的な利益があることを宣言します。

ここで我々 は、翻訳、再現性のある、柔軟なモデルの作成に多くの利点を持つ糖尿病慢性創傷でバイオ フィルムの研究のための新しいマウスモデルをについて説明します。
最初の技術革新は、ヘアレス マウスの使用です。糖尿病の慢性創傷治癒の10、11を勉強する他のマウスのモデルが開発されているが、すべてはワックス?...

バイオ フィルムの慢性の傷1の解像度が低い要因として強調表示されている高分子物質のマトリックスに埋め込まれた微生物の複雑なコミュニティであります。これらの高度に組織化された、永続的な微生物集団の研究は、四肢周辺変質的な知覚メカニズムに血行不良が検出されない病変2につながる糖尿病の患者のため特に重要です。アメリカ合衆国では、糖尿病患者の 15% は自分たちの生活の中に、少なくとも 1 つの潰瘍を開発すると推定されます。これは無量の感情的、社会的負担を述べない治療3,4で約 280 億ドルの経済的な支出に変換します。傷のベッドで癒しのイベントでこれらのバイオ フィルムに与える影響を永続化する微生物ができる要因を理解することは、影響を受ける患者のためのより良いケアを駆動し、新しい処置のアプローチの開発を推進することが不可欠です。したがって、再現で変換可能な体内細菌のホストの相互作用の探索にモデルの確立が重要です。
慢性の傷でバイオ フィルムの影響を研究する、マウスモデルを正常に開発されています。ただし、これらのモデルは、しばしば、髪の種を利用し、犠牲にされた動物が、時間がかかり、高価なから摘出組織の細菌細胞のプレート カウントによってバイオ フィルム クリアランスを評価します。
感染症の評価における動物の終了点サンプリング バイオフォトニック代替最初、Contagらにより提案されました。(1995 年)5,人は抗生物質の治療効果を測定する恒常発光サルモネラから発光をキャプチャする手法を開発しました。他の生物発光発光細菌の活用の研究が続いた。たとえば、Rochettaら。(2001 年)6検証を激化させた電荷結合素子を用いた発光を測定することにより大腸菌太もも感染症のマウスを研究する感染症モデルおよび後で、Kadurugamuwaら。(2003 年)7は黄色ブドウ球菌マウスにおけるカテーテル治癒モデルにおけるいくつかの抗生物質の有効性を調査するための設計されたひずみの性質を発光の光子を利用しました。
ここで特徴づけられるメソッドを提示ヘアレス マウスの糖尿病を誘発する、制作し膿の前もって形成された発光バイオ フィルムで傷を予防接種、バイオフォトニック感染のモニタリングを実施する簡単なプロトコル生体内でイメージ投射システムを使用しています。直通快速、その場で、慢性の傷でバイオ フィルムを評価する非侵襲的かつ定量的プロセスを提供していますさらに、詳細な分析により、癒しの顕微鏡イメージング傷、断続的な採血のためなどサイトカイン測定と組織学のためのターミナルの組織コレクション。

<table><tbody><tr><td>Opsite</td><td>Smith &amp;amp; Nephew</td><td>Model 66000041</td><td>Smith &amp;amp; Nephew Flexfix Opsite Transparent Adhesive Film Roll 4&quot; x 11yards</td></tr><tr><td>SKH-1 mice &lt;strong&gt;Crl:SKH1-Hr&lt;/strong&gt;&lt;sup&gt;hr&lt;/sup&gt;</td><td>Charles River Breeding Laboratories</td><td>SKH1</td><td>Hairless mice, 8 weeks old</td></tr><tr><td>Streptozotocin (STZ)</td><td>Sigma Aldrich</td><td>S0130-1G</td><td>Streptozocin powder, 1g</td></tr><tr><td>AccuChek glucometer</td><td>Accu-Chek Roche</td><td>Art No. 05046025001</td><td>ACCU-CHEK CompactPlus Diabetes Monitoring Care Kit</td></tr><tr><td>Pseudomonas aeruginosa Xen 41</td><td>Perkin Elmer</td><td>119229</td><td>Bioluminescent Pseudomonas aeruginosa</td></tr><tr><td>Polycarbonate membrane filters</td><td>Sigma Aldrich</td><td>P9199</td><td>Millipore polycarbonate membrane filters with 0.2 &amp;mu;m pore size</td></tr><tr><td>Dulbelcco phosphate buffer saline (DPBS)</td><td>Sigma Aldrich</td><td>D8537</td><td>PBS</td></tr><tr><td>Tryptic soy agar</td><td>Sigma Aldrich</td><td>22091</td><td>Culture agar</td></tr><tr><td>Meloxicam</td><td>Henry Schein Animal Health</td><td>49755</td><td>Eloxiject (Meloxicam) 5mg/mL, solution for injection</td></tr><tr><td>10% povidone-iodine (Betadine)</td><td>Purdue Products LP</td><td>301879-OA</td><td>Swabstick, Betadine Solution. Antiseptic. Individ. Wrapped, 200/case</td></tr><tr><td>4% paraformaldehyde</td><td>Fisher Scientific</td><td>AAJ61899AK</td><td>Alfa Aesar Paraformaldehyde, 4% in PBS</td></tr><tr><td>Capillary glass tube</td><td>Fisher Scientific</td><td>22-362-566</td><td>Heparinized Micro-Hematocrit Capillary Tubes</td></tr><tr><td>Silicone to make splints</td><td>Invitrogen Life Technologies Corp</td><td>P-18178</td><td>Press-to-Seal Silicone Sheet, 13cm x 18cm, 0.5mm thick, set of 5 sheets</td></tr><tr><td>Tryptic soy broth</td><td>Sigma Aldrich</td><td>22092</td><td>Culture broth</td></tr><tr><td>IVIS Spectrum</td><td>Perkin Elmer</td><td>124262</td><td>In vivo imaging system</td></tr><tr><td>IVIS Spectrum Isolation chamber</td><td>Perkin Elmer</td><td>123997</td><td>XIC-3 animal isolation chamber</td></tr><tr><td>HEPA filter</td><td>Teleflex</td><td>28022</td><td>Gibeck ISO-Gard HEPA Light number 28022</td></tr><tr><td>Biopsy punches</td><td>VWR International Inc</td><td>21909-142</td><td>Disposable Biopsy Punch, 5mm, Sterile, pack of 50.</td></tr><tr><td>Biopsy punches</td><td>VWR International Inc</td><td>21909-140</td><td>Disposable Biopsy Punch, 4mm, Sterile, pack of 50.</td></tr><tr><td>Glucose</td><td>J.T.Baker</td><td>1916-01</td><td>Dextrose, Anhydrous, Powder</td></tr><tr><td>Citric acid</td><td>Sigma Aldrich</td><td>C2404-100G</td><td>Citric Acid</td></tr><tr><td>Mastisol</td><td>Eloquest Healthcare</td><td>HRI 0496-0523-48</td><td>Mastisol Medical Liquid Adhesive 2/3 mL vial, box of 48</td></tr><tr><td>Corning 96-well black plates</td><td>Fisher Scientific</td><td>07-200-567</td><td>96-well clear bottom black polysterene microplates</td></tr><tr><td>25 gauge 5/8 inch needle</td><td>BD</td><td>305122</td><td>Regular bevel needle</td></tr><tr><td>Bransonic M Ultrasonic Cleaning Bath</td><td>Branson Ultrasonics</td><td>N/A</td><td>Ultrasonic Cleaner</td></tr></tbody></table>

come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model

慢性の傷、特に糖尿病患者での構造化されたバイオ フィルムとしての細菌の存在は、創傷治癒や解像度を防ぐためと考えられます。慢性マウスモデルの傷は、微生物とホストの間の根本的な相互作用を理解するために使用されています。これまで開発したモデルは、髪の動物の使用は、細菌による傷組織のターミナル ・ コレクションに依存します。これらのモデルに重要な洞察を得た、この実験動物の大きい数が必要です、サンプリングは時間がかかります。慢性創傷におけるバイオ フィルムの進行を評価するいくつかの最適な革新を組み込んだ新規マウスモデルを開発した:) それは毛の取り外しのための必要性を排除、ヘアレス マウスを利用してb) 永続化の即時評価とホストにこれらのコミュニティの効果を可能にする傷に前もって形成されたバイオ フィルムを適用されます。c) 遺伝子組み換え生物発光菌緑膿菌、感染症の研究ごとに必要な動物の数を減らすをリアルタイムで監視できるように、光を定量化することでバイオ フィルムの進行を監視します。このモデルで単一の全深さの傷は STZ 誘発糖尿病マウスの裏面に生産、膿発光ひずみ Xen 41 のバイオ フィルムを接種しました。傷からの光出力生体内イメージング システムに、生体内でその場で急速なバイオ フィルムの可視化と傷内バイオ フィルム細菌の局在に毎日記録されます。この手法は柔軟な遺伝子組み換え種、複数種バイオ フィルムなど、他の微生物を調査する使用することができ、抗菌閉塞性ドレッシングを含む抗バイオ フィルム戦略をテストで特別な価値があります。

この新しいモデルを開発する c57bl/6 j マウスを過去に使用した上毛 SKH 1 の活用において多くの利点が見られました。通常 STZ 注射を受ける動物糖尿病の発症と緩やかな減量を経験します。ただし、傷の癒しの実験以前による所内ダンらによって提示されたモデルを再現します。(2012 年)9 c57bl/6 j、急激な体重減少を使用していた (...

Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

光側に来る: In Vivo糖尿病ヘアレス マウス モデルにおける慢性創傷における緑膿菌バイオ フィルム感染症の監視

ここで我々 はリアルタイム、非侵襲的発光緑膿菌のバイオ フィルム創傷感染の監視のヘアレス マウスを用いた新規糖尿病マウスモデルをについて説明します。このメソッドは、他の菌種と複数種バイオ フィルムを含む、遺伝子組換え微生物の感染症を評価および antibiofilm 戦略の有効性をテストして合わせることができます。

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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Research

JoVE Journal

Immunology and Infection

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

13.1K ビュー.  Michigan State University. ここで我々 はリアルタイム、非侵襲的発光緑膿菌のバイオ フィルム創傷感染の監視のヘアレス マウスを用いた新規糖尿病マウスモデルをについて説明します。このメソッドは、他の菌種と複数種バイオ フィルムを含む、遺伝子組換え微生物の感染症を評価および antibiofilm 戦略の有効性をテストして合わせることができます。

ビデオ: Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

フローセル内の緑膿菌と出芽酵母のバイオフィルム

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

免疫・感染学

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Murine Model of Wound Healing

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

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

創傷治癒のマウスモデル

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

医学

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

生物発光イメージングが続くマウス皮下モデルにおける医学関連の異物にカンジダ·アルビカンスバイオフィルム発展

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性緑膿菌創傷感染の遅延接種モデル

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

後部皮下ポリビニルアルコールスポンジの移植と切除テール皮膚創傷モデルを用いた急性創傷治癒の評価

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

糖尿病マウスの慢性創傷を作成するためのプロトコル

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Assessing Biofilm Dispersal in Murine Wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

ネズミの創傷におけるバイオフィルム分散の評価

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

バイオフィルム感染熱傷創傷研究のための長期ブタモデルの開発

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

著者スポットライト:創傷バイオフィルム形成における宿主-微生物相互作用の研究 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

バイオフィルム感染と不可視創傷のブタモデル

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

光側に来る: In Vivo糖尿病ヘアレスマウスモデルにおける慢性創傷における緑膿菌バイオフィルム感染症の監視

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さらに動画を探す

この動画の章

フローセル内の緑膿菌と出芽酵母のバイオフィルム

のRNAの単離緑膿菌マウス消化管に定着

創傷治癒のマウスモデル

生物発光イメージングが続くマウス皮下モデルにおける医学関連の異物にカンジダ·アルビカンスバイオフィルム発展

慢性緑膿菌創傷感染の遅延接種モデル

後部皮下ポリビニルアルコールスポンジの移植と切除テール皮膚創傷モデルを用いた急性創傷治癒の評価

糖尿病マウスの慢性創傷を作成するためのプロトコル

ネズミの創傷におけるバイオフィルム分散の評価

バイオフィルム感染と不可視創傷のブタモデル

バイオフィルム感染と不可視創傷のブタモデル

光側に来る: In Vivo糖尿病ヘアレス マウス モデルにおける慢性創傷における緑膿菌バイオ フィルム感染症の監視

要約

さらに動画を探す

この動画の章

フローセル内の緑膿菌と出芽酵母のバイオフィルム

のRNAの単離緑膿菌マウス消化管に定着

創傷治癒のマウスモデル

生物発光イメージングが続くマウス皮下モデルにおける医学関連の異物にカンジダ·アルビカンスバイオフィルム発展

慢性緑膿菌創傷感染の遅延接種モデル

後部皮下ポリビニルアルコールスポンジの移植と切除テール皮膚創傷モデルを用いた急性創傷治癒の評価

糖尿病マウスの慢性創傷を作成するためのプロトコル

ネズミの創傷におけるバイオフィルム分散の評価

バイオフィルム感染と不可視創傷のブタモデル

バイオフィルム感染と不可視創傷のブタモデル

光側に来る: In Vivo糖尿病ヘアレスマウスモデルにおける慢性創傷における緑膿菌バイオフィルム感染症の監視