この作品は、英国心臓財団 (PG/13/60/30406) およびバーミンガムの大学によって支えられました。

動物のすべてのプロシージャは、動物福祉倫理的な調査機関および英国ホーム オフィス (イギリス、プロジェクト ライセンス 40/3745) によって承認されました。C57BL/6 背景、8-12 週齢、体重 19 25 g、両方の性別の上でマウスを使用します。
1. 動物の麻酔
<ol>
	<li>誘導室にマウスを置き、5% 100% 酸素とイソフルランの混合物によって麻酔を誘導します。使用マウスは完全に移動を停止するまでの 0.5 L/min の流量を待つと呼吸の頻度は稀になります。</li>
	<li>商工会議所からマウスを外し、電気クリッパーと腹部を剃る。腹腔内の汚染を避けるために可能な限り徹底的に腹部から髪を削除します。</li>
	<li>仰臥位でマウスを置き、マウスの鼻に麻酔システムにリンクされている円錐を配置します。2-3% (正確な割合が動物から動物を異なる場合) にイソフルランの割合が低下します。動物は寝ているその呼吸あえぎ通常、回避よりも低くされていることを確認します。あえぎが発生した場合、0.5% イソフルランの割合を減らします。
	<ol>
		<li>適切な anesthetization を確認し、それが否定的な場合にのみ手術を開始するペダルの反射を確認します。乾燥を防ぐため目に獣医軟膏を適用します。</li>
	</ol>
	</li>
	<li>適用抗殺菌液 (1% ヒビテン) マウスの腹部とワイプ滅菌綿を使用して領域は、手術部位 (例えば、内側から循環的に外側へ) の潜在的な再汚染を除く方法で芽します。3 回繰り返します。</li>
</ol>
2. 下大静脈狭窄とマウスの回復のアプリケーション
注: すべての楽器同様 (綿棒、ガーゼ、生理食塩水等) の材料として手術領域の接触を滅菌する必要があります。オペレーターは、手術前にスクラブし、プロシージャの間に滅菌手袋とガウンを着用する必要があります。顕微鏡のハンドルは、滅菌アルミホイルでラップされます。
<ol>
	<li>手術前と全体の実験期間中に、マウス抗痛み治療を与えます。たとえば、ブプレノルフィン 0.1 mg/kg 皮下 30 分前に手術、その後 12 時間毎まで動物を安楽死を使用します。この方法では血栓症は無菌性炎症に同様に成長するとは、麻酔前投薬として抗炎症薬を使用しないでください。</li>
	<li>滅菌ドレープとマウスをカバーし、外科サイトを公開するドレープに穴をあけます。はさみを使って、腹部正中線、胸骨からダウン 1.5 cm に沿って切開を行います。綿棒を使用して、マウスの左側に腸を外在、乾燥を避けるために暖かい (37 ° C) を生理食塩水に浸した滅菌ガーゼで覆います。</li>
	<li>下大静脈と左腎静脈と下大静脈の融合のサイトから尾側方向の側枝 (場合があります 0、1、またはそれらの 2) を特定します。として可能な限り下大静脈に近い不活性プロレン縫合を 7-0 のすべての目に見える側面枝を縛る。
	<ol>
		<li>鉗子と側枝ではなく、直接片手に鉗子とそれらを囲む脂肪質のティッシュを持ってそれらを引き出すし、一方で鉗子で枝の下にトンネルを作るを保持しないでください。背面枝は閉じないでください。</li>
	</ol>
	</li>
	<li>鉗子で周囲の組織を持ち、プルを下大静脈と左と下大静脈、大動脈正確下大静脈と左腎静脈の間の角度で小さい区域の張力を作り出します。下大静脈も 2-3 mm 以下 (尾側方向) から大動脈を区切ることは事実上不可能であることを考慮します。</li>
	<li>右手を大動脈と下大静脈の穴鉗子先端の左右逆の動きを使用してください。覚えておいてより多くの動きを作った、容器の損傷の可能性が高く。理想的には、3 - 5 の動きの数を最小化しようとします。</li>
	<li>約 2 cm の長さの 7-0 不活性プロレン縫合の部分を準備します。演算子の左側下大静脈に近くでマウスの腹腔内にそれを配置します。</li>
	<li>下大静脈と左もう一度プルします。下大静脈の反対側に、ヒントが表示される大動脈と下大静脈間の穴にあなたの右の鉗子の先端を挿入します。貫通穴 (図 3A) 縫合糸とそれを引き戻すを取る。</li>
	<li>縫合糸で仮結び目を作るが、終了しません。結び目にフックとして曲がって 30 G 針 (「スペーサー」) を配置して今閉じます (図 3B)。</li>
	<li>スペーサー (図 3C) を削除します。</li>
	<li>綿と腹腔内に腸の芽、それ均等にそれらを配布を返します。</li>
	<li>連続的なループを使用して 6-0 vicryl の縫合糸で腹膜を閉じます。結び目として最初と最後のループを閉じます。</li>
	<li>金属のステープルを使用して近い皮。</li>
	<li>マウスを暖かい (37 ° C) の 0.5 ml 注入有機物の液体のバランスを回復する皮下グルコースを生理食塩水。</li>
	<li>暖かい (25 ° C) 商工会議所や部屋で個々 のケージにマウスを置くし、食べ物を入れてケージの中の水をゲルします。マウスは完全に (通常、約 1 h) 回復までを観察します。</li>
</ol>

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著者が明らかに何もありません。

ここでは、血流歪み、深部静脈血栓症の主要なトリガー因子を模倣した下大静脈狭窄のプロトコルが表示されます。48 時間以内 c57bl/6 マウスの 65-100%、25-50 %2-6 h6,8,23内でマウスの血栓症、下大静脈狭窄を誘導する (ブリル、未発表データ、2016)。メソッドの主要な制限は、血栓のサイズ24 (図 4)、短期 (6 h) と長期 (48 h) 下大静脈狭窄後観察の変動です。そのような変動の理由 (ことを考えると、スペーサーなど、条件の同じ麻酔等が使用される) あいまいなままですが、1 つはその解剖学的推測可能性がありますマウス (例えば、幅下大静脈、数、サイドとバックの両方の位置の間の変化枝) この現象の根底にあります。血栓サイズの可変性は主な結果血栓症有病率 (血栓マウスの %) をなります。血栓症の有病率は、分割表とフィッシャーの正確確率検定を使用して比較できます。例外は、ポドプラニン抑制抗体の注射が7が観察された後に減らされた血栓が同じ血栓症有病率とサイズ実験の 1 つでした。
このメソッドは、静脈血栓症の開始を検討するときに便利です。それは血栓症に最終的につながる細胞の募集など、血管壁の初期イベントを調査することができます。薬のプロまたは反 thrombotic 効果は、プライマリの読み出しをされている血栓症有病率とこのモデルを使用して評価できます。48 h の狭窄は、するプロトロンビンの表現型が予想される場合、6 h 狭窄を使用できるに対し、抗血栓性の表現型を明らかにするのに適用されます。血栓と周囲の下大静脈壁の組織学的解析を行うもできます。
結紮または特許左サイドの枝かどうかすべき質問は開いたままになります。1 つのグループは、側枝の結紮は血栓のサイズを増加せず、側枝下大静脈結紮サイトに 1.5 mm 厚よりも近くの場所は劇的に血栓開発25を損なうことを示しています。側枝の閉鎖可能性がありますそれらの血管内皮細胞障害を誘発しても手術26の時間を増やします。私たちの手で側枝の閉鎖の欠如大幅に減少 (48 h 狭窄後 10-30% に血栓症の有病率ブリル、未発表)、したがって我々 はすべての目に見える側面枝を縛る。
理想的には、マウス、同じ背景にも、さまざまなソースから可能性がありますがあるわずかに異なる血栓症有病率としては、濾胞を使用ください。(たとえば、生化学的な) 任意のパラメーターに DVT 自体の影響を検討すると、偽手術動物が使わなければなりません。Sham 群は、同じ手術を受けるが、下大静脈を結紮が緩く閉じられ、狭窄を生じることがなく残っています。
このプロトコルの最も頻繁な間違い (重要なステップ) 大動脈、それは出血の結果は正確に若干低くはなりますが、船舶間の角度で下大静脈を分離する努力であります。大量の出血が発生すると、マウスの良い回復なりそう、実験を停止し、動物を安楽死させるをお勧め。通常、マウスも回復、ケージ内を移動し、重量を大幅に失うことはありません。男性と女性の両方の 20 グラム以上のマウスを使用してお勧めし、戦闘および傷害を避けるために実験終了時まで手術後個々 のケージに動物 (特に男性) を維持します。それは、雄マウスが女性27より大きい血栓を作り出す深部静脈血栓症の別の (電解) モデルで報告されています。我々 のデータの分析は雌雄マウス (ブリル、未発表) の血栓症有病率の相当な違いを発表していません。したがって、研究者は、男女両方のマウスで下大静脈狭窄症モデルを用いて実験を行うよう奨励されます。
縫合やステープルを解き明かすことが否定できない特に長い実験 (週そして長い)。このように、マウスは縫合の整合性のために特別 1 日少なくとも 2 回チェックする必要があり、血のトレース、床敷の外観に特別な注意を与えられるべき。
その他の動物のモデルのような下大静脈狭窄は人間への翻訳の面で限界があることに留意。たとえば、人間の深部静脈血栓症は静脈弁内部に生じるに対し、マウス IVCs にはバルブはありません。また、人間は、静脈の血流の重要なメカニズム加速されて筋肉ポンプと脊髄縦書きをあります。対照的に、マウス脊髄横向きには役割の心臓に血液を返すを支える筋肉ポンプがあります。ひと疾患へのマウスのデータを変換するとき、これらの制限を考慮する必要があります。

深部静脈血栓症 (DVT) 深部静脈血栓症の開発は、通常 (しかしだけでなく) の足。肺塞栓症と組み合わせて (PE; 一緒に静脈血栓塞栓症、静脈血栓塞栓症として示される) それは年間約 90万人で開発し、深刻な健康問題と経済問題を抱えて1,2を表します。PE、深部静脈血栓症の合併症は、血栓が元の場所から戸建を取得し、呼吸不全と死を引き起こすことができる肺に達するときに発生します。静脈血栓塞栓症による死者数は、エイズ、乳房癌や交通事故、結合された3から死亡率を超えています。
以外にも深部静脈血栓症を引き起こす主要な要因の理由を知られている癌や外傷など、移動4、5の欠如です。手術 (特に整形外科)、麻痺、長距離路線やその他の理由からこれがあります。静脈の血流は筋ポンプに依存し、したがって血液の停滞で手足固定結果フロー静脈弁の血栓症に 。記載方法の目的は、このような血液流れ歪み6,7を要約することです。下大静脈 (IVC) の流量制限人間血栓6の構造のような人間の静脈弁の血栓の形成の結果作成された条件を模倣しています。血栓の大部分は赤く、赤い血球、フィブリン、血小板の結合で構成されています。血栓は、小さな「ザンのライン」を人間静脈血栓で説明に似ている (図 1)、血小板の濃縮「白い部分」を持っています。血栓の両方の部分含むも好中球8、将来血栓6,9サイトで募集する最初の細胞の間であります。赤い部分の好中球は、白い部分に好中球網8は欠けているように見えるに対し好中球細胞外トラップ (網) を放出します。同様に人間の深部静脈血栓症にマウスでの血栓症は血中 D ダイマー レベル (図 2) を伴います。低分子量ヘパリン (エノキサパリン) 患者における深部静脈血栓症予防のため使用はまたマウスの血栓症を防ぐ。この方法の重要な利点は、人間の深部静脈血栓症10の特徴である内皮侵食9の不在です。この機能により、下大静脈狭窄、血栓症の誘導などよりも深部静脈血栓症のより臨床的に関連するモデル塩化第二鉄、内皮細胞の侵食を誘導して血栓が血小板11,の主に成っている12,13。下大静脈完全な停滞のモデルは、いくつかのチーム14,,1516が優先されます。狭窄、容器、維持は残差流と対照をなしてうっ滞のアプリケーションは流れが完全に停止し、こうして血栓症サイトに全身投与物質のアクセシビリティを制限します。また、狭窄やうっ滞による血栓症のメカニズムが異なっているようだ: 狭窄により局所炎症の開発 (内皮、Weibel Palade 本体のコンテンツのリリース、免疫細胞の活性化と血管壁への血小板) うっ滞ではなく血栓症を誘発するようであるに対し"immunothrombosis"6,9,17の原因に基づいて、特に、組織因子とその他の凝固、線溶機構18,19,20。したがって、狭窄症、うっ血のモデルは、その機構は確かに重なる可能性がありますが、静脈血栓開発のわずかに異なる側面を反映してください。DVT21,22の電解 IVC モデル (EIM) は、血管壁を部分的にのみ遮蔽血栓を誘導します。したがって、さまざまな薬物の全身投与の血栓の成長に及ぼす影響をテストするために便利です。ただし、このモデルには、この機構の病態生理学的関連性の少なくとも議論の余地を作って電流による下大静脈壁の保全 (針の挿入) の中断と血栓症の誘導が想定しています。

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stenosis of the inferior vena cava: a murine model of deep vein thrombosis

深い静脈の血栓症 (DVT) とその壊滅的な合併症、肺塞栓症、高い死亡率に深刻な健康問題があります。静脈血栓形成のメカニズムは曖昧のまま。(例えば手術や長距離飛行後) 流動性の欠如は、深部静脈血栓症につながる主な要因の 1 つです。流動性の欠如の病態生理学的結果は、静脈弁の血流停滞です。ここでは、モデル、血栓症運転要因としてこのような流れの妨害を模倣するを説明します。このモデルでは、流量制限 (狭窄) 下大静脈 (IVC) が作成されます。48 h の IVC 内腔の約 90% の閉鎖血栓人間のそれらに構造的に類似の開発で起因します。類似: i) 血栓検索ボリュームのほとんどは赤、すなわち、iii) 裸内皮化単分子膜、iv) 血中 D ダイマー レベル、および v) 可能性を防ぐために赤血球とフィブリン、ii) 白い部分 (ザーンの行) の存在から成ります低分子ヘパリンによる血栓症です。制限には血栓と実際の変数のサイズが含まれている野生型マウスの一定割合 (0 - 35%) 血栓をもたらさないことがあります。目視および計測のほか血栓は血栓性の動態は、超音波検査などの非侵襲的技術によって視覚化が可能します。イベント (例えば血管壁細胞の募集) を直接観察に適用可能性があります短い時間ポイント (1-6 h)、生体顕微鏡血栓形成の前します。世界中の複数のチームによるこのメソッドの使用は、深部静脈血栓症発生の基本的なメカニズムを明らかにし、その予防のために有益かもしれない潜在的なターゲットを識別する可能になりました。

ここ、下大静脈の偏流による深部静脈血栓症のモデルを説明します。下大静脈狭窄症の誘導が歪みを開始し、(この場合は 48 h) でしばらくして血流の停滞による血栓、人間に似たような構造の DVT の開発血栓 (図 1A)。下大静脈内血栓の存在は視覚的に容易に検出できる (図 1B)。モデルと人間の深部静脈血栓症の重要な類似は血中 D ダイマー レベル (図 2) です。フィブリン分解産物である D ダイマーと血でのプレゼンス アクティブな血栓性プロセスが起こっていることを示唆しています。
モデルは図 3の Cのステップバイ ステップが表示されます。下大静脈は慎重に公開、下大静脈と大動脈の間の穴をあけて、縫合 (プロレン 7-0) が下大静脈の下にある穴から引っ張られます。[スペーサーが削除されます後スペーサー (30 G 針、図 3D)、上、下大静脈を縫合糸で結紮します。この手順により、約 90% の残りの 10% の特許を残して IVC 内腔の閉鎖。これにより最終的に血栓症につながる血流停滞であります。
図 4では、変数血栓サイズ モデルの主要な欠点を示しています。これらの血栓が同じ実験で得られたすべては同じ起源、年齢と同じような体重の男性マウスを野生型で実行されます。塞栓 (血栓症有病率 100%) を生産マウスのすべてのサイズは明らかに広い範囲で異なります。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56697/56697fig1.jpg"> 
図 1:48 時間制限/狭窄後の代表的な血栓。(A) 典型的な血栓後 48 h 下大静脈狭窄。(赤血球濃縮) 赤と白 (血小板濃縮) 部品に注意してください。(左) と (B) を下大静脈 (右) 血栓の有無。スケール バー = 2 mm.<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56697/56697fig2.jpg"> 
図 2: 下大静脈狭窄後の D ダイマー上昇します。マウスは、下大静脈狭窄に服従しました。血液が示された時点で取られた、遠心分離 (2,300 × g、5 分) によって準備された 3.8% クエン酸ナトリウム、プラズマを安定化 1:9、D-ダイマー濃度は、製造元の指示に従って ELISA キットによって定められました。サークル指定 sham 群、十字架は、下大静脈狭窄を指定します。<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56697/56697fig3.jpg"> 
図 3:マウスにおける DVT の下大静脈 (IVC) モデルの制限/狭窄症の流れ。モデルの連続段階が掲載されています。(A) 下大静脈と大動脈の間の穴をあけて、下大静脈の周りそれを縫合糸は引っ張られます。(B) 結び目はスペーサー (30 G 針) で閉じられます。(C)スペーサーを削除: マウスを閉じる前に外科医を見て最終的なビュー。黄色の破線は、下大静脈を設定します。LRV は、左腎静脈を略します。スケール バー = 0.2 mm。 (D) スペーサー (30 G 針)。スケール バー = 2 mm.<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56697/56697fig4.jpg"> 
図 4.血栓のサイズの変動。 マウスは、48 h 下大静脈狭窄に服従しました。血栓が同じ実験で得られたが提示します。スケール バー 2 mm.<a href="//ecsource.jove.com/files/ftp_upload/56697/56697fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis at JoVE.com

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

私たちは深部静脈血栓症のマウスモデルとして下大静脈狭窄をここで記述します。このモデルは、人間の静脈血栓症の主要なトリガーの 1 つ、血流制限を繰り返します。

下大静脈狭窄: 深部静脈血栓症のマウスモデル

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

stenosis-inferior-vena-cava-murine-model-deep-vein

Research

JoVE Journal

Immunology and Infection

23.7K ビュー.  University of Birmingham. 私たちは深部静脈血栓症のマウスモデルとして下大静脈狭窄をここで記述します。このモデルは、人間の静脈血栓症の主要なトリガーの 1 つ、血流制限を繰り返します。

ビデオ: Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

免疫・感染学

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 ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

細菌性前立腺炎のマウスモデルにおける接触性アロディニアの測定

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

高周波超音波による監視マウスのうっ滞による深部静脈血栓症

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深い皮膚注入として、モデルカンジダ皮膚感染症病理組織学的解析のための

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

生体内でマウスにおける活性酸素種のイメージング創モデル

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

リンパ管内皮細胞のフローサイトのマウス肝臓の消化

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

口腔好中球評価のためのマウス歯肉炎の堅牢な合字誘発モデル

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...