現在の研究は、厚生助成金R01-HL092188、R01-DK083385とR01HL098294と、HKEとDFG（ドイツ学術振興）研究フェローシップ（に麻酔教育研究（FAER）助成財団の米国国立研究所によってサポートされていましたGR2121/1- 1）および米国心臓協会グラントAGへ。

一般的な発言。すべての操作は、直立解剖顕微鏡（ライカ）の下や手術凝固装置を用いて実行する必要があります。実験とシャム群のマウスは、結果の比較可能性を確保するために年齢と体重で一致する必要があります。体温、血圧、麻酔と流体管理は、安定した全体の実験中に監視する必要があります。 1。麻酔と手術<ol><li> C57BL / 6マウスは、10から16週齢の範囲内でなければなりません、彼らは性別をマッチさせたそれぞれのグループでなければなりません。 </li><li>ペントバルビタールナトリウム（70 mg / kg体重のIP）にマウスを麻酔し、約麻酔を維持しています。は10 mg / kg / hのペントバルビタールナトリウム。過量投与は有意に血圧を下げることができるので、結果を変更することができます。 </li><li>サーマル·フィードバックに接続された直腸の温度計のプローブを用いた温度制御加熱されたテーブル（RT、Effenberg、ミュンヘン、ドイツ）の上に置いてマウス37℃で体温を維持するためにコントローラ℃、 </li><li>取り外し可能なテープでテーブルに取り付けられた上下肢で、手術のテーブルの上に仰臥位にマウスを置きます。 </li><li>生存実験髪のクリッピングを含む適切な無菌テクニックを、ヨードフォア（例えばBetadine）で皮膚の消毒、オートクレーブ滅菌した器具の取扱いの場合に、外科医のための滅菌手袋が必要とされるマスクを使用します。 </li></ol> 2。ポータルトライアド咬合<ol><li>皮膚をカットするため、マウスの毛アレルギーのリスクを防止することにより、マウスの毛の吸入を防ぐためにミネラルオイルを切開領域をカバーしています。あなたが容易に肝臓を公開できるように、最大​​の上端に白線の正中開腹切開を行います。可能であれば、出血を防ぐために、凝固電極（エルベ、ICC50、チュービンゲン、ドイツ）で切開を行います。 </li><li>尾が方向であることの位置にマウスを使って加熱テーブルを配置外科医にテッド。その位置は、トライアドをローカライズし、縫合糸を配置するために最適な設定を提供します。 </li><li>ポータルトライアドを公開するために湿った綿棒の綿棒を使用して、右側の腸を置換する。 </li><li>少しダイアフラムに向かって中央と左葉と腸に向かって右葉をシフトします。それはあなたの右、左/中央ローブの分岐上にポータルトライアドのクリアな視界が得られます。 </li><li>慎重にポータルトライアドを検査し、当たり障りのないピンセットでそっと右葉を保持します。それはあなたの左のポータルトライアドの素敵なビューを提供します。 </li><li>左から右側に肝動脈、肝静脈、胆管を含む左側のポータルトライアドの下でニードルホルダーを介して、縫合糸（エチコン、ノルダーシュテット、ドイツ7/0ナイロン縫合糸）を一度視覚的に特定された場所に針をマウスの。 </li><li>ステッチは、トライアドの下に密接にする必要があります。深いステッチの下に配置され静脈VACA劣っを傷つけることができるトライアド。したがって、ステッチを配置する前に下大静脈を識別するために、ピンセットで肝右葉を押したままにします。 </li><li>鉗子で右側の針の先端を取り、肝臓を傷つけることなく肝臓領域外にそっと引き出します。 </li><li>縫合エッペンドルフチュー​​ブ（水で満たされた約3グラム）の各端部に接続し、両方の縫合糸は、縫合糸ホルダーを介して終了に配置します。縫合糸は、右のトライアドに干渉しないようにステッチの遠位縫合糸ホルダーを配置します。 </li><li>ウェイトを適用することにより、トライアドは、肝臓の左と中央葉への血液供給の中断を引き起こし、即座に閉塞されます。成功した閉塞は容易に虚血葉の淡い白化の目視検査によって確認することができる。ぶら下がって重みが極から放出されると肝臓が灌流されたときとは対照的に、色の変更はすぐに消えます。この部分肝虚血モデルではblooを維持することで腸間膜混雑を回避する肝右葉にdフロー。 </li><li>腹腔内に腸を交換してください。 37.0で水に浸した濡れた綿棒℃で、肝臓と腸を暖かく保つさらに、手術を終えた後、市販の食品用ラップでマウスを覆う体温が安定して維持する。 </li><li>実験プロトコルごとに、必要に応じて虚血を実行します。定義されている虚血時間のリリース後に中央値と肝左葉の重量と再灌流は直ちに開始されます。この手法は、マウスで可逆的虚血を作り出すことができます。それ以上の虚血時間が実行されません場合、慎重に縫合糸を除去します。 </li><li> 15分前に直接再灌流後の腹腔内に約400μlの塩化ナトリウムを追加します。再灌流時に流体の損失を補償する100μlの量は30分ごとに流体を代用し続けています。 </li><li>生存実験のために手術創は連続筋肉壁の縫合、皮膚wiを使って閉じている4/0縫合番目。 </li></ol> 3。肝灌流の変化以下の結果のパラメータは肝虚血後に肝障害を決定するために推奨されています。 肝酵素（例えば、ALT、AST）および肝臓組織学（H＆E染色）は、以下の肝虚血再灌流3,9を決定します 。 <img alt="図1" src="/files/ftp_upload/2550/2550fig1.jpg" /> 図1肝虚血にかける重みシステムのモデル：吊り下げ重量システムは、左と中央葉3の肝虚血を誘発するために設立されました。 （）したがって、尾状核と右葉はそっと左葉から分離した。右葉は明らかに右、中央と左葉の分岐上にポータルトライアドを表示するには、その後わずかにシフトした。 8.0ナイロン縫合糸は、左肝トライアド下に置かれた。 （B）それぞれの縫合糸の端にある小さな重量（3グラムエッペンドルフTU水で満たされたBES）が付属して適用した。肝虚血は、赤から淡い左と中央葉の色の変化によって表示されます。この実験的なデザインは、重量負荷を適用し、解放することによって肝左トライアドの正確な閉塞および再潅流することができます。 <img alt="図2" src="/files/ftp_upload/2550/2550fig2.jpg" /> 図2アラニン（ALT）とアスパラギン酸（AST）マウスにおけるアミノトランスフェラーゼの異なる肝虚血時間の影響。部分的なポータルトライアドの虚血は、示された（ - 60分虚血0）として誘導した。再灌流2時間後、ALT（A）とA​​ST（B）を測定した。 7匹/群 - 結果は平均±SE 6のように表される。 0分虚血（偽コントロール）と比較* P &lt;0.05。

<ol>
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利害の衝突が宣言されません。

本研究では左肝ポータルトライアドの閉塞にかけるウェイトシステムを使用してマウスモデルにおける虚血を行う手法を説明しています。それは学ぶことが簡単な方法ですが、次の点を考慮する必要があります。初めに非常によくある間違いは、ポータルトライアドと下大静脈を含む深いステッチです。呼吸や心拍​​数が増加し抵抗とマウスの早期死亡の結果心臓に血液を戻すの減少により15分以内に遅くなります。下大静脈の含むを防ぐための最善の方法は、鉗子で右の耳たぶを押したままにしてステッチする前に下大静脈をローカライズすることである。もう一つの重要な問題は、重みがまた右葉への血流を停止し、左右のポータル裁判に過度のストレスになることが重いのではないということです。したがって、エッペンドルフバイアル中の水の量は、マウスの重量とsに調整する必要がありますutureホルダーは、ステッチの場所から遠位に配置する必要があります。ウェイトは低血圧にある場合、反対側を完全に停止されていません。両方ともミスは簡単には、それぞれ中央と左葉右葉または不十分な色の変化の淡いへの追加色の変化で検出されています。したがって、ウェイトを適用後に肝色の変化は10分ごとに制御する必要があります。別の一般的な手術の合併症は、針で肝臓の負傷です。したがって、右の葉は、針の先端が容易に見られ、ニードルホルダーで撮影することができるように当たり障りのない鉗子で押したままにする必要があります。初めにこれらの落とし穴を考慮している場合は肝虚血にかける体重のシステムは、ポータルトライアドのクランプに関連付けられている肝臓の損傷のばらつきを最小化することにより、虚血による再現性の高い傷害を提供しています。壁掛け重量システムを使用して、ポータルのトライアドは、全体を通して一度に操作される外科手術、肝葉に大幅に少ないダメージを与える。さらに、全く肝臓や腸の混雑は、この手法では発生しません。多くの組織の外傷、特に虚血プレコンディショニングの間のような複数の虚血サイクル中にクランプの再適用して、血管クランプを削除し、置き換えることによって発生します。クランプの再適用による肝葉の操作に起因する組織損傷を防止しながら、我々の観察によると、肝臓組織から離れた場所にある重みを掛けるの使用は、信頼性の高い咬合の利点を提供しています。一緒に、本研究では、クランプに関連付けられている変動と制限を最小限に抑え、虚血時の門脈三管閉塞のためにぶら下げ軽量システムの実現可能性を提供しています。したがって、この手法は、肝虚血を研究して検討する他の研究者のために興味があるかもしれません。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ペントバルビタールナトリウム（致命的プラス） </td><td> Vortech製薬Lsは、（株） </td><td> VPL 9372 </td><td>生理食塩水で4 mg / mLの</td></tr><tr><td>縫合糸、プロレン8.0 </td><td>エチコン、米国</td><td> M8739 </td><td>再利用可能な</td></tr><tr><td>解剖顕微鏡</td><td>ライカ</td><td> N / A </td><td>寛大な作動距離を考慮して</td></tr><tr><td>暖房表</td><td> RT、エッフェンベルガー、ドイツ</td><td> N / A </td><td>のみと単一のプロバイダ</td></tr><tr><td>手術器具</td><td> WPI、デュモン</td><td></td><td>鉗子、ニードルホルダー</td></tr><tr><td>凝固剤</td><td>エルベ、ICC50、チュービンゲン、ドイツ</td><td> N / A </td><td> coagulatorsの異なるプロバイダ</td></tr></tbody></table> 表1特異的試薬および装置。

use of a hanging-weight system for liver ischemia in mice

虚血による急性肝障害は、いくつかの臨床試験の手続きなどの肝移植、肝腫瘍の切除や外傷の修理時に発生する可能性があると高い死亡率1-2を持つ肝臓に障害が発生することができます。したがって、肝虚血のマウスの研究では、3月9日薬理学的および遺伝学的研究を活用する機会を提供することにより、研究の重要な分野となっています。具体的には、遺伝子の組織特異的欠失（CRE、FLOXシステム）条件付きマウスは、特定の組織10月13日のタンパク質の役割についての洞察を提供しています。ので、手動でマウスのポータルトライアドをクランプに関連付けられている技術的な難しさから、我々は以前に3に記載されているポータルトライアド閉塞用壁掛け重量システムを使用して、体系的な評価を行った。壁掛け重量システムを使用して、我々はまたので、肝葉の損傷を引き起こすことなく、ポータルトライアドの左側の枝の周囲に縫合糸を配置する利用可能な最高のクランプため血管に肝組織の密接な場所の肝組織の損傷を引き起こす可能性があります。さらに、肝トライアドの右枝はまだ右肝葉への血流が維持されるようには腸の混雑は、この手法では発生しませんので、灌流されています。さらに、ポータルトライアドは、全体の手術を通して一度に操作されています。その結果、プレコンディショニングのような手順では、虚血再灌流の短い時間で、簡単に行うことができます。アラニン（ALT）とアスパラギン酸（AST）アミノトランスフェラーゼの血清レベル3,9によって測定された別の虚血再灌流時間を行うことにより、このモデルの体系的評価は、肝臓の損傷による肝虚血時間の密接な相関関係を明らかにした。肝虚血再灌流障害と断続的なハングし体重のシステムを使用する場合は一緒になって、これらの研究は、再現性の高い肝臓の損傷を確認します。したがって、この手法は他の研究者の関心のために役に立つかもしれませんマウスの肝虚血研究におけるED。したがって、ビデオクリップは、この手法の詳細なステップバイステップの説明を提供します。

Watch this Scientific Journal Video about Use of a Hanging-weight System for Liver Ischemia in Mice at JoVE.com

Use of a Hanging-weight System for Liver Ischemia in Mice

我々は、ポータルトライアド閉塞のためにぶら下がって体重システムの新規マウスモデルを確立した。この手法は、マウス肝モデルにおける虚血の今後の調査のための役に立つかもしれません。

マウスの肝臓虚血のためにハンギング軽量システムの利用

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

use-of-a-hanging-weight-system-for-liver-ischemia-in-mice

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15.9K ビュー.  University of Colorado, Denver. 我々は、ポータルトライアド閉塞のためにぶら下がって体重システムの新規マウスモデルを確立した。この手法は、マウス肝モデルにおける虚血の今後の調査のための役に立つかもしれません。

ビデオ: Use of a Hanging-weight System for Liver Ischemia in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

マウスにおける冠動脈閉塞のために掛かる重量のシステムの使用

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

医学

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

隔離された腎動脈閉塞のためのハンギングウェイトシステムの利用

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

新しいハーブ治療法を評価するために敗血症の動物モデルの使用

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

微妙な子宮内膜症の術中検出：腹膜整合性の喪失に伴う骨盤痛の検出および治療のための新たなパラダイム

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

マウスの脊髄損傷のモデルとしての光血栓誘発性局所虚血

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

マウスやラットのための低フローデジタル麻酔システムの利用

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

表面パッド心電図を使用したマウスにおける心筋虚血および再灌流障害の確認

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

実験的炎症性腸病気のマウスモデルで使用するため、腸管へのホーミングを増加するヒト間葉系幹細胞の超音波ガイド下心臓内注射

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

肝臓生物学のEx Vivoモデルとしてのマウス精密カット肝臓スライス

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...