著者らはニコZantl、クラウスプフェッファー、とベルンハルトホルツマンに感謝。この研究はドイツ学術振興、ボン - バートゴーデスベルク、ドイツ（; Voが450/10-1 GRK - 840）によってサポートされていました。

1。マウスの準備<ol><li>麻薬液の腹腔内注射によってマウスを麻酔（試薬の表を参照）と仰臥位に入れてください。 </li><li>マウスの足は運転中に動物の安定した位置を確保するためにプレートにテープで固定する必要があります。 </li></ol> 2。動作<ol><li>一般的にヒトに静脈内注射に使用されるIVカニューレを、取る、そしてその先端からプラスチック製のチューブ循環2mmを彫る。我々は、死亡率（; 16GA（1,7 x45mm、180 mL /分）14GA（2,0 x45mm、270 mL /分）BD Venflon 18GA（1,2 x45mm、80 mL /分））を制御するためのカニューレの3種類のサイズを使用。 </li><li>腹部の皮膚を徹底的に消毒した後、15ミリ程度の正中線に沿って切開する。白線に沿って腹部の筋肉と腹膜を切開による腹膜の洞窟を開きます。 </li><li>盲腸ポールを特定し、慎重に盲腸、回腸末端と綿棒を使用することによって腹部から上行結腸を引き出します。 </li><li>回盲弁から15mm遠位、一つは穴を開けるために7 / 0縫合糸と上行結腸の壁を持っています。これにより、腸の血管の病変は回避する必要があります。縫合糸は、2つの手術ノットで大腸壁に固定されています。 </li><li>今穿刺7 / 0縫合糸から近準備カニューレ1〜2ミリメートルと上行結腸。プラスチックチューブの溝が漿膜とレベルになるまで慎重にコロンにカニューレを挿入する。 </li><li>カニューレの周囲7 / 0縫合糸の自由端を入れ、正確にプラスチック製のチューブの準備畝に2倍の結び目を置きます。 </li><li>今、それはコロンのantimesenteric壁を通って7 / 0縫合糸とステッチの針を取る。連続して、2つの手術の結び目は、さらに、大腸壁にプラスチックチューブを固定するために実行する必要があります。縫合糸の端をカット。 </li><li>カニューレはほとんどの鉄の一部を取得し、固定7 / 0縫合糸にプラスチック製のチューブに近いオフ（1ミリメートル）をカット。 </li><li>今一便の小さな先端はステントの上部に表示されるまで慎重に綿棒を使用することにより、大腸ステントに向かって盲腸から便を牛乳に持っています。 </li><li>生理食塩水0.5 mLを腹腔内投与による腹膜洞窟とperforme流体の蘇生に腸を戻す。 </li><li>連続縫合（4 / 0）と腹膜を閉じます。 </li><li>特異縫合（4 / 0）で皮膚を閉じます。 </li></ol> 3。術後処置<ol><li>十分な食料と水を含んでいるはずのケージに動物の背部を、置く。鎮痛のために、強力な鎮痛物質（我々はブプレノルフィンを使用）の腹腔内投与は定期的に実行する必要があります。 </li><li>操作後の最初の2日以内に、人は動物を6時間ごとに制御する必要があります。 </li></ol> 4。シャムCASP <ol><li>ステップ1.1から2.4までを実行します。 </li><li>穿刺コロンをしないでください。徒歩での2.6から2.8を実行してください。 </li><li>手順2.10から3.2までを実行します。 </li></ol> 5。スピー<ol><li> CASPは14Gカニューレの以下の手順で1.1から3.1を使用して実行</li><li> CASP後の5時間は、1つは、再び動物を操作する必要があります。ステップ1.1から1.2にしたがって、再度マウスを準備し、麻酔</li><li>腹壁の縫合糸を開きます。 </li><li>挿入されたステントで上行結腸を引き出します。 </li><li>慎重にステントを固定する縫合糸をカットし、ステントを削除します。 </li><li>単一の反転縫合（7 / 0）とコロンの欠陥を閉じます。 </li><li>腹腔内に戻って腸を入れ、生理食塩水10mLで2回、後者をフラッシュする。 </li><li>連続縫合（4 / 0）と腹膜を閉じます。 </li><li>特異縫合（4 / 0）で皮膚を閉じます。 </li><li>ステップ3.1から3.2に従ってください</li></ol> 6。代表的な結果操作の後数時間以内に、動物は最初の敗血症の臨床症状を示しています。病気の典型的な症状は発汗、モビリティ、剛毛で覆われたコートを削減している、食物摂取、体重の損失とも減少休暇の動作を減少させた。連続して全身感染症を伴う重症腹膜炎を開発する動物は、通常48時間以内に死亡する。挿入されたステントのサイズに応じて、個別の死亡率を生成することができます。 100％の死亡率で14Gステント結果、16Gは、70％の死亡率を与え、50％の死亡率は、18Gステント（図1a）を使用してアクセスすることができます。 100％の生存率におけるシャムの操作の結果。通常、すべての動物は、CASP後の最初の48時間わたってるしき死ぬ。従って、この時点で生きているすべての動物は生存者とみなすことができる。しかし、我々は稀な&quot;遅い死&quot;CASP後を検出するために少なくとも72時間の追加の観察期間をお勧めします。 スピーの結果は、ステントが削除されたCASP後の期間に依存します。 45％の生存率のCASP結果の後に介入3H。ステントは、CASP、10％だけ後5hに削除される場合動物は、手順を生き残る。 100％（図1b）の死亡率の9H結果の後にステント除去。シャムスピーは100％の生存率につながる。通常後でCASP後48時間以上死ぬ動物が、あるため、スピーの設定については、私たちは10日間の観察期間をお勧めします。 浮腫、血管拡張、erythematic腸壁、パイエル板の鋭い境界、腸管麻痺、無料の流体、および敗血症性分泌（図2）：24時間CASP後の腹部内臓の肉眼検査では、腹膜炎の典型的な兆候を示しています。 全身感染症は、細菌学的分析によって示すことができる。 12時間CASP後、大規模な細菌の量が腹腔洗浄液中で検出することができる、血液、肝臓、肺、脾臓、および腎臓。細菌の定性分析は、E.のような典型的な腸内細菌による全身感染症を明らかに大腸菌 、バクテロイデス属、腸球菌種、など（図3）。 CASP誘発性敗血症は、プロと抗炎症性サイトカインやケモカインの地元とも全身のリリースで現れます。 TNF、IL -1β、IL - 6、IL - 10、MCP - 1などのCASP、かなりの量の後12時間はならず、臓器の懸濁液、例えば肝臓、肺、脾臓、腎臓の上清中に血液中のELISA法により測定することができます。 （図4）。 あなたの実験室でCASPを確立するときに、生存率、細菌およびサイトカインの放出は、CASPの適切なパフォーマンスを得るための制御パラメータとして解釈されるべきである。標準はすべての実験の設定に対して、システムを読み出すように後で、これらのパラメータを取得することもできます。 <img src="/files/ftp_upload/2299/2299fig1.jpg" alt="図1" /> 図1a：CASP後の生存 。 CASPの手術後の生存率は、挿入ステントの直径に依存する。死亡率の100％の割合、70％の死亡率で16G CASPの結果、および18G CASPで14G CASPの結果は、50％の死亡率につながる。シャムCASPは100％の生存率に関連付けられています。 。N = 20/group B：生存率スピー手術後のステントの除去の時間に依存する。 14G CASP後の3Hでのステントの除去は55％の死亡率につながる。 100％の死亡率で14G CASPの結果の後に9Hでのステントの除去に対し、90％の死亡率で14G CASPの結果の後5時間でのステントの除去。シャムスピー（5H）100％の生存率の結果。 N = 10/group（シャムスピー、スピー9H）、N = 20/group（3Hスピー、スピー5H）。 <img src="/files/ftp_upload/2299/2299fig2.jpg" alt="図2" /> 図2：CASP後の腹部内臓の24時間 。腹壁はそれぞれ、16​​G CASPまたは偽CASP後24削除されました。偽手術のマウスの内臓は生理的に見えます。対照的に、浮腫、充血、dilatated腸、およびパイエル板の分界のCASPの結果は、重度の腹膜炎を実証。 <img src="/files/ftp_upload/2299/2299fig3.jpg" alt="図3" /> 図3：細菌学 。 CASPは、手術後12時間以内に腸内細菌によって、いくつかのコンパートメントや臓器の重篤な感染症につながる。細菌数はコロニー形成単位（cfu）1mLあたり、それぞれとして与えられる。偽手術したマウスから採取した細菌培養（図示せず）が完全に無菌である。 16G CASPは、n = 5/group。 <img src="/files/ftp_upload/2299/2299fig4.jpg" alt="図4" /> 図4：サイトカイン 。いくつかのサイトカインやケモカインは、臓器の上清においても血漿中で検出可能であるととしてCASPは、全身免疫応答をトリガします。図は、例示的に炎症性サイトカイン腫瘍壊死因子（TNF）とインターロイキン-6（IL - 6）だけでなく、主に抗炎症IL - 10を示しています。 CASP後の12Hは、注目に値するレベルは血漿、肝臓、肺、および脾臓で検出することができます。しかし、IL - 10は、CASP後の肺と脾臓で検出することはできません。偽手術は、どのサイトカインの分泌（図示せず）につながるものではない。 16G CASPは、n = 5/group。

<ol>
	<li>Maier, S., Traeger, T., Entleutner, M., Westerholt, A., Kleist, B., Huser, N., Holzmann, B., Stier, A., Pfeffer, K., Heidecke, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture+versus+colon+ascendens+stent+peritonitis:+two+distinct+animal+models+for+polymicrobial+sepsis.">Cecal ligation and puncture versus colon ascendens stent peritonitis: two distinct animal models for polymicrobial sepsis.</a> Shock. 21, 505-511 (2004).</li><li>Bougaki, M., Searles, R. J., Kida, K., Yu, J. D. e., Buys, E. 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利害の衝突は宣言されません。

、CASPは小動物で行うことができます簡単に標準のパラメータにより監視するために、高い再現性であり、優れた機能が実現可能であることを読み出す提供、そして密接にの臨床場面を模倣している：CASPは、貴重な敗血症モデルに対して想定されるほとんどの基準を満たしている腹部の敗血症の複数菌モデルです。腹部敗血症。比較研究では、盲腸結紮穿刺（CLP）がで腹腔内膿瘍形成のモデルを明らかにする一方、CASPは、早期かつ着実に増加して全身感染症や炎症（全身性炎症反応症候群）とびまん性腹膜炎につながることを示している可能性が持続し、マイナー全身性炎症の徴候（1）。スピーの技術は、ヒトにおいて最も重要な治療原則である連続した外科フォーカス衛生、と腹膜炎の臨床経過をシミュレートする可能性を提示。 CASPは、CASPのモデルを使用して敗血症の異なる主題に関する出版物の選定免疫学、薬理学、外科、集中治療医学、等例えば、敗血症や腹膜炎の周りすべての質問に対して適用され、したがって、敗血症の研究を扱うすべての分野に有用であることができる（1-14）を以下に示します。 CASPで始まるとき、人はいくつかの練習が正確にかつ迅速に操作を実行するために必要であることを認識する必要があります。手術の経験が役に立つかもしれませんが、必須ではありません。我々は、CASPの適切なパフォーマンスのためのパラメータとしてletalityの検出をお勧めします。大規模なステントは、薄いものより扱いやすいように一つは14G CASPで始まる必要があります。訓練されたCASPの外科医は10分以内つのマウスを操作することができます。どんな実験を開始する前に、あなたがその期間に近いことを確認する必要があります。あなたが手術といくつかの経験を得たとき、あなたはあなたのスキルをチェックするために生存運動を実施する必要があります。 14G CASPは、100％の致死性になるはずです。我々の研究室での初心者は、彼らが実験を開始する前に14G CASPと100％の致死率を生成することができることを証明する必要があります。また、細菌学およびサイトカインの測定は、敗血症を確認するために実行する必要があります。あなたがCASPの顕微ステップと始まりの問題がある場合は、拡大鏡や手術用顕微鏡の使用が役立ちます。しかし主に、CASPは、よくこれらのユーティリティなしで行うことができます。私たちの生存率の再現のために、それは同じ材料、特にステント（BD Venflon、下記参照）が、使用されることは絶対に必要です。そうでなければ、結果のかなりのバリエーションが可能です。さらに、我々は、例えば、上の外科医の個性に応じて結果の違いがあることを示唆している。しかし、そのような要因の影響は小さいはずです。 要約では、CASPは密接に腹部敗血症の臨床的状況を模倣する実用的な、シンプルで、再現性と貴重な敗血症モデルです。したがって、敗血症の研究のすべてのフィールドに適しています。

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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Narcotic fluid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 0.5 mL Rompun with 4.0 mL Ketanest, and add 5.5 mL of saline solution (0.9%). 
For complete anesthesia, inject intraperitoneally 10 &mu;L/g body weight</td>
</tr>
<tr>
<td>Rompun (20 mg/ml Xylazine)</td>
<td>&nbsp;</td>
<td>Bayer AG 51368 Leverkusen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketanest (25mg/ml Esketamine)</td>
<td>&nbsp;</td>
<td>Pfizer Pharma GmbH, 10785 Berlin, Germany</td>
<td>647028001E</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 18GA (1,2x45mm, 80ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391457</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 16GA (1,7x45mm, 180ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391455</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 14GA (2,0x45mm, 270ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391456</td>
<td>&nbsp;</td>
<tr>
<td>Catgut Polyester 4/0 white, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>17218113</td>
<td>&nbsp;</td>
<tr>
<td>Mariderm black 7/0, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>18104900</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colon ascendens stent peritonitis (casp) - a standardized model for polymicrobial abdominal sepsis

敗血症は、世界中の集中治療室での永続的な問題として残っている。敗血症の複雑なメカニズムを理解することは、この分野で新しい治療法を確立するための前提条件となります。従って、動物モデルは、密接に人間の病気を模倣しても十分に科学的な質問に対処することができることが必要です。コロンはステント性腹膜炎Ascendens（CASP）げっ歯類における複数菌腹部敗血症のための高度に標準化されたモデルです。このモデルでは、小型のステ​​ントは、外科的に腹腔内に腸内細菌の連続的な漏洩につながるマウスやラットの上行結腸に挿入されます。手続きの腸内細菌による腹膜炎、全身菌血症、臓器の感染症の結果、およびいくつかの炎症と抗炎症性サイトカインの全身だけでなく、ローカルのリリース。 CASPの致死率は、挿入されたステントの直径によって制御することができます。このモデルの変形、介入（スピー）を持つ、いわゆるCASPは、臨床実践における一般的な手順に従って、再手術による感染病巣を除去する機会を発生させます。 CASPは、密接に腹部敗血症の臨床経過を模倣容易に学習可能な、再現性の高いモデルです。それは、免疫学、感染症学、または手術など、いくつかの科学分野での質問で勉強する方法をリードしています。

Watch this Scientific Journal Video about Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis at JoVE.com

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

コロンはステント性腹膜炎Ascendens（CASP）げっ歯類における複数菌腹部敗血症のための高度に標準化されたモデルです。この記事では、CASPの外科的手順を説明します。 CASPのモデルおよびその変種は、敗血症の主題に関わる様々な問題の体系的調査を可能にする。

コロンは（CASP）腹膜炎ステントAscendens - 複数菌腹部敗血症のための標準モデルを

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

colon-ascendens-stent-peritonitis-casp-standardized-model-for

Research

JoVE Journal

Immunology and Infection

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

19.3K ビュー.  University of Greifswald. コロンはステント性腹膜炎Ascendens（CASP）げっ歯類における複数菌腹部敗血症のための高度に標準化されたモデルです。この記事では、CASPの外科的手順を説明します。 CASPのモデルおよびその変種は、敗血症の主題に関わる様々な問題の体系的調査を可能にする。

ビデオ: Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

マウスにおけるオートファジーを研究するためのモデルとして、盲腸結紮穿刺誘発性敗血症

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

免疫・感染学

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生児モデル敗血症マウスの制御モデル

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

敗血症誘発免疫抑制のセカルライゲーションと穿刺および鼻腔感染の二重モデルの設計

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部大動脈瘤マウスモデルにおける脈拍伝播速度、分解性およびひずみの測定

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

グラム陰性細菌敗血症の新生児画像モデル

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

マウス真菌敗血症モデルにおける効率的な尾静脈注射のための現代的な温暖化/抑制装置

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

標準化腸ループモデルを用いたマウスにおける腸透過性と好中球経上皮移動の機能評価

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

マウスにおける サルモネラ ・チフス菌誘発性敗血症性腹膜炎の特性評価

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

敗血症の盲腸結紮および穿刺誘発マウスモデルにおける信頼性の高いバイオマーカーの評価

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

免疫学的洞察のための強化された腸内チッププラットフォーム

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

著者スポットライト:感染症研究のための免疫適格臓器チップモデルの開発

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

腸粘膜免疫応答を解析するための免疫適格腸内チップモデル

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...