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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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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18 (4), 593-603 (1995).</li><li>Rocchetta, H. L., 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=Validation+of+a+noninvasive,+real-time+imaging+technology+using+bioluminescent+Escherichia+coli+in+the+neutropenic+mouse+thigh+model+of+infection.">Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.</a> Antimicrob Agents Chemother. 45 (1), 129-137 (2001).</li><li>Kadurugamuwa, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+treatment+modulates+the+host+immune+system+to+enhance+Pseudomonas+aeruginosa+wound+biofilms.">Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.</a> Infect Immun. 82 (1), 92-100 (2014).</li></ol>

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 &#38; Nephew</td>
			<td>Model 66000041</td>
			<td>Smith &#38; Nephew Flexfix Opsite Transparent Adhesive Film Roll 4&#34; x 11yards</td>
		</tr>
		<tr>
			<td>SKH-1 mice Crl:SKH1-Hrhr</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 &#956;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. ...

come-to-light-side-vivo-monitoring-pseudomonas-aeruginosa-biofilm

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

緑膿菌誘発肺傷害モデル

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

微生物群集を学びます<em&gt;インビボ</em&gt;：ホスト間の媒介の相互作用のモデル<em&gt;カンジダ</em&gt;と<em&gt;緑膿菌</em&gt;航空で

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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 ...

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

移植片対宿主病の誘導および

に対する抗生物質の有効性をテストするための人工痰培地の使用

長期的な慢性

緑膿菌誘発肺傷害モデル

微生物群集を学びます

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

慢性緑膿菌創傷感染の遅延接種モデル

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

移植片対宿主病の誘導および

に対する抗生物質の有効性をテストするための人工痰培地の使用

長期的な慢性

緑膿菌誘発肺傷害モデル

微生物群集を学びます

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

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