この研究は、ヘルスリサーチおよびゲノムCanada.JSのカナダの協会によってサポートされていたがアルバータ遺産Foundationfor医学研究、心臓とCanadaandの脳卒中財団カナダ糖尿病協会から給与を支援賞を保持しています。シアラーの実験室には、この手順を教えるデビッドワッサーマンと氷楽Bracyに感謝。

<ol>
	<li>Halseth, A. E., Bracy, D. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+to+basal+and+insulin-stimulated+skeletal+muscle+glucose+uptake+in+the+high-fat-fed+rat.">Limitations to basal and insulin-stimulated skeletal muscle glucose uptake in the high-fat-fed rat.</a> Am J Physiol Endocrinol Metab. 279, E1064-E1071 (2000).</li><li>Halseth, A. E., Bracy, D. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+to+muscle+glucose+uptake+due+to+high+fat+feeding.">Limitations to muscle glucose uptake due to high fat feeding.</a> Am. J. Physiol. 279, E1064-E1071 (2000).</li><li>Muniyappa, R., Lee, S., Chen, H., Quon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+for+assessing+insulin+sensitivity+and+resistance+in+vivo:+advantages,+limitations,+and+appropriate+usage.">Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage.</a> Am J Physiol Endocrinol Metab. 294, 15-26 (2008).</li><li>Cho, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+a+diabetes+mellitus-like+syndrome+in+mice+lacking+the+protein+kinase+Akt2+(PKB+beta).">Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).</a> Science. 292, 1728-1731 (2001).</li><li>Dubois, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SHP-1+protein+tyrosine+phosphatase+negatively+modulates+glucose+homeostasis.">The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis.</a> Nat Med. 12, 549-556 (2006).</li><li>Pacini, G., Thomaseth, K., Ahren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+to+glucose+tolerance+of+insulin-independent+vs.+insulin-dependent+mechanisms+in+mice.">Contribution to glucose tolerance of insulin-independent vs. insulin-dependent mechanisms in mice.</a> Am J Physiol Endocrinol Metab. 281, 693-703 (2001).</li><li>Herbach, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant-negative+effects+of+a+novel+mutated+Ins2+allele+causes+early-onset+diabetes+and+severe+beta-cell+loss+in+Munich+Ins2C95S+mutant+mice.">Dominant-negative effects of a novel mutated Ins2 allele causes early-onset diabetes and severe beta-cell loss in Munich Ins2C95S mutant mice.</a> Diabetes. 56, 1268-1276 (2007).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+insulin+resistance+in+mice+lacking+adiponectin/ACRP30.">Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.</a> Nat Med. 8, 731-737 (2002).</li><li>Potenza, M. A., Marasciulo, F. L., Tarquinio, M., Quon, M. J., Montagnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+spontaneously+hypertensive+rats+with+rosiglitazone+and/or+enalapril+restores+balance+between+vasodilator+and+vasoconstrictor+actions+of+insulin+with+simultaneous+improvement+in+hypertension+and+insulin+resistance.">Treatment of spontaneously hypertensive rats with rosiglitazone and/or enalapril restores balance between vasodilator and vasoconstrictor actions of insulin with simultaneous improvement in hypertension and insulin resistance.</a> Diabetes. 55, 3594-3603 (2006).</li><li>Ayala, J. E., Bracy, D. P., McGuinness, O. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+the+design+of+hyperinsulinemic-euglycemic+clamps+in+the+conscious+mouse.">Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse.</a> Diabetes. 55, 390-397 (2006).</li><li>Wasserman, D. H., Ayala, J. E., McGuinness, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation.">Lost in translation.</a> Diabetes. 58, 1947-1950 (2009).</li></ol>

手順は、カルガリー動物ケアの大学および使用委員会によって承認され、実験用の実験動物科学のガイドラインについては、カナダの協会を遵守した。著者らは利害の対立または開示はありません。

最初にヒトのインスリン感受性の調査のために開発、クランプの手順は、現在、実験用ラットやマウスなど他の種に適応されています。インスリン抵抗性の調査を動物モデルでは、インスリン感受性と関連する病理の病態生理を理解するだけでなく、臨床的価値の1,2を持っている治 ​​療的介入を特定するのに重要な援助を提供します。動物のインスリン感受性を評価するいくつかのメソッドは、3を採用されている。このような技術は、ブドウ糖負荷試験のバリエーション（GTTs）およびインスリン負荷試験（ITTS）4-6と同様に空腹時の定常状態7-9由来のインスリン感受性/抵抗のインデックスが含まれています。しかし、インスリン感受性がプライマリ実験目標と技術的実現可能性のときにインスリンクランプを制限していないのは、標準のまま。ここでは、意識、無拘束のラットにおける最も基本的なクランプの手順を示します。変数外因性グルコースが血糖を維持するために注入されながら、インスリンが一定の速度で投与される。動物の循環へのアクセスは、動脈と頚静脈カテーテルの注入によって得られる。動物のストレス（ハンドリング）と麻酔がグルコースの流束とインスリン感受性を変化させるとして、これらの要因を最小化するモデルは、この手順では好まれて利用されている。 インスリンクランプは、一般的に糖尿病、心血管系および薬学研究に使用されています。手順への変更は、同位体トレーサー（放射性または安定）の付加を含む。これらは、調査員が特定の組織や生化学的経路がクランプに対応して変更される方法を決定できるようにするという点で強力​​です。一般的な同位体が2の使用を含む - [14C] -デオキシグルコース全身や組織特異的グルコース処理だけでなく、グルコースの流束の測定のための[3H] -グルコースを検討する。方法論およびレポート課 ​​題の優れたレビューワッサーマンらによるインスリンクランプの手順を実行する。10,11を強くお勧めします。

<table border="1"><tbody><tr valign="top"><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr valign="top"><td> Intramedicポリエチレンチューブ（PE - 50） </td><td>フィッシャーサイエンティフィック</td><td> 14から170 - 12B </td><td> 0.58ミリメートル（0.023&quot;）0.965ミリメートルのX外径（0.038&quot;）の内径</td></tr><tr valign="top"><td>ダウコーニングシラスティックラボチューブ</td><td>フィッシャーサイエンティフィック</td><td> 11から189 - 15C </td><td> 0.76ミリメートル（0.030&quot;）の内部直径1.65ミリメートルのX外径（0.065&quot;） </td></tr><tr valign="top"><td>タイゴンS - 50 - HLメディカルチューブ</td><td>ハーバード装置</td><td> PY2 72から1251 </td><td> 3.2ミリメートルの内径（0.125&quot;）4.7ミリメートルのX外径（0.1875&quot;） </td></tr><tr valign="top"><td>ロックタイトスーパー接着剤</td><td>グランド＆玩具</td><td> 32237 </td><td>ゲルコントロール</td></tr><tr valign="top"><td>無菌手術ブレード</td><td> VWR </td><td> BD371610 </td><td></td></tr><tr valign="top"><td>湾曲したマイクロ解剖鉗子</td><td>ジョージティーマンアンドカンパニー</td><td> 160から20 </td><td> × 2 </td></tr><tr valign="top"><td>ストレートマイクロ解剖鉗子</td><td>ジョージティーマンアンドカンパニー</td><td> 160から15 </td><td> × 2 </td></tr><tr valign="top"><td>湾曲した止血剤</td><td>ジョージティーマンアンドカンパニー</td><td> 105-1135 </td><td> × 2 </td></tr><tr valign="top"><td>ストレート止血剤</td><td>ジョージティーマンアンドカンパニー</td><td> 105-1130 </td><td> × 2 </td></tr><tr valign="top"><td>止血鉗子の先端ガード</td><td>ロビンスインスツルメンツ株式会社</td><td> 15.09-2-004 </td><td></td></tr><tr valign="top"><td>ストレート外科用はさみ</td><td>ジョージティーマンアンドカンパニー</td><td> 105から402 </td><td></td></tr><tr valign="top"><td> VENOJECTマルチサンプルルアーアダプター</td><td>テルモメディカルプロダクツ</td><td> 810127A </td><td> 21ゲージ、1インチ</td></tr><tr valign="top"><td>滅菌カテーテルイントロデューサー</td><td>ベクトンディッキンソン</td><td> 406999 </td><td></td></tr><tr valign="top"><td> 14ゲージのブラントニードル</td><td>ベクトンディッキンソン</td><td> 511310 </td><td> 14ゲージ、2インチ</td></tr><tr valign="top"><td>滅菌縫合糸</td><td>ジョンソン＆ジョンソンメディカルプロダクツ</td><td> 1679H </td><td>シルク、サイズ3〜0 </td></tr><tr valign="top"><td>非滅菌外科用縫合糸</td><td>アンジオテックファーマシューティカルズ</td><td> SP116 </td><td>シルク、サイズ4から0 </td></tr><tr valign="top"><td>綿棒</td><td> VWR </td><td> 10806-005 </td><td></td></tr><tr valign="top"><td> 4plyガーゼパッド</td><td> VWR </td><td> CA43845 - 062 </td><td></td></tr><tr valign="top"><td>小動物コードレスクリッパーズ</td><td>ハーバード装置</td><td> 729063 </td><td></td></tr><tr valign="top"><td>イソフルレン</td><td>ハロカーボン製品株式会社</td><td> IPN - 45 </td><td></td></tr><tr valign="top"><td>麻酔カート</td><td>ベンソン医療産業、（株） </td><td></td><td></td></tr><tr valign="top"><td> 70％エタノール</td><td>フィッシャーサイエンティフィック</td><td> HC - 1000 </td><td></td></tr><tr valign="top"><td> Betadineの消毒液</td><td>西洋医薬品流通センター株式会社</td><td> 105267 </td><td></td></tr><tr valign="top"><td>モデル11 Plusのシリンジポンプ</td><td>ハーバード装置</td><td> 702208 </td><td></td></tr><tr valign="top"><td>ステンレス鋼チューブカプラ</td><td>ハーバード装置</td><td> 72から4434 </td><td> 23ゲージ、インチ0.3 </td></tr><tr valign="top"><td>ステンレス鋼チューブのプラグ</td><td>ハーバード装置</td><td> 72から4436 </td><td> 23ゲージ、0.5インチ</td></tr><tr valign="top"><td>ステンレス鋼ブラント針</td><td>インステックラボラトリーズ、（株） </td><td> LS22 </td><td> 22ゲージ</td></tr><tr valign="top"><td> 60 ° Y -コネクタ</td><td>スモールパーツ</td><td> STCY - 22から05 </td><td> 22ゲージ</td></tr><tr valign="top"><td> CritSpinマイクロヘマトクリット遠心機</td><td>アイリスのサンプル処理</td><td> CS12 </td><td></td></tr><tr valign="top"><td>ミニ遠心機</td><td>フィッシャーサイエンティフィック</td><td> 05-090-100 </td><td></td></tr><tr valign="top"><td>マイクロ遠心チューブ</td><td> VWR </td><td> 53550-778 </td><td></td></tr><tr valign="top"><td> 50ミリリットルポリプロピレン遠心チューブ</td><td> VWR</td><td> 89004-364 </td><td></td></tr><tr valign="top"><td> 1ミリリットルプラスチックスリップのヒントシリンジ</td><td>ベクトンディッキンソン</td><td> 309602 </td><td></td></tr><tr valign="top"><td> 3ミリリットルプラスチックLuerlokヒントシリンジ</td><td>ベクトンディッキンソン</td><td> 309585 </td><td></td></tr><tr valign="top"><td>ヘパリン抗凝固剤の注入</td><td>西洋医薬品流通センター株式会社</td><td> 102824 </td><td>メーカー：LEO製薬株式会社コンク。 1000 IU </td></tr><tr valign="top"><td> EDTA溶液</td><td>プロメガ株式会社</td><td> V4231 </td><td> 0.5 M、pH8.0の</td></tr><tr valign="top"><td>生理食塩水</td><td>西洋医薬品流通センター株式会社</td><td> ABB7983154 </td><td>メーカー：ホスピーラ0.9％塩化ナトリウム</td></tr><tr valign="top"><td> 50％のブドウ糖</td><td> Vetoquinol </td><td> 8DEX012D </td><td></td></tr><tr valign="top"><td>ヒューマリン- R </td><td>イーライリリー</td><td> HI - 210 </td><td> 100U/ml </td></tr><tr valign="top"><td> 1ミリリットルインスリン用注射筒</td><td>ベクトンディッキンソン</td><td> 309311 </td><td></td></tr><tr valign="top"><td> Fisherbrand * Hematoシールシーラント</td><td>フィッシャーサイエンティフィック</td><td> 02から678 </td><td></td></tr><tr valign="top"><td> Fisherbrand * Microhematocritキャピラリーチューブ</td><td>フィッシャーサイエンティフィック</td><td> 22-362-574 </td><td></td></tr><tr valign="top"><td>ワンタッチウルトラテストストリップ</td><td> LifeScan、（株） </td><td> AW 085 - 314H </td><td></td></tr><tr valign="top"><td>ワンタッチウルトラ血糖測定器</td><td> LifeScan、（株） </td><td> AW 085 - 314B </td><td></td></tr><tr valign="top"><td> Pentobarbitolナトリウム</td><td>チェバサンテAnimale </td><td> 1715 138 </td><td>コンク。 54.7mg/ml </td></tr><tr valign="top"><td>レッド研究所ラベリングテープ</td><td> VWR </td><td> 89097-932 </td><td></td></tr><tr valign="top"><td>青い研究所ラベリングテープ</td><td> VWR </td><td> 89097-936 </td><td></td></tr><tr valign="top"><td>計量器</td><td>フィッシャーサイエンティフィック</td><td> 01-913-88 </td><td></td></tr><tr valign="top"><td>渦</td><td> VWR </td><td> 58815-234 </td><td></td></tr><tr valign="top"><td>タイマー</td><td> VWR </td><td> 62344-641 </td><td></td></tr></tbody></table>

hyperinsulinemic-euglycemic clamp in the conscious rat

2型糖尿病（T2D）が急速に罹患率が高まっています。不十分なインスリンの生産や高血糖のレベルでインスリン生産、T2D結果を利用することができないことのいずれかによって特徴付け。インスリン感受性を評価する&quot;ゴールドスタンダード&quot;は高インスリン血症、正常血糖クランプまたはインスリンクランプです。この手順では、インスリンは血糖値の低下、その結果一定の速度で注入する。一定のレベルで血糖値を維持するために、外因性グルコース（D50）は、静脈循環に注入される。恒常性を維持するために注入されたグルコースの量は、インスリン感受性の指標である。ここで、我々は慢性的にカテーテルを挿入、無拘束、覚醒ラットにおける基本的なクランプの手順を示しています。このモデルでは、血液が動物に最小限のストレスで収集することができます。麻酔の誘導後、正中切開が行われ、左総頸動脈と右頸静脈はカテーテルをされています。挿入されたカテーテルは、体外と確保、ヘパリン生理食塩水でフラッシュされます。動物を毎日監視体重増加で、実験の前に4〜5日で回復されている。手術前のレベルに体重を取り戻すだけの動物が実験に使用されています。実験当日、ラットを絶食させるとインスリンとD50を含むポンプに接続されています。ベースラインのグルコースは、動脈ラインから評価し、実験（血糖値）全体でベンチマークを使用されています。これに続いて、インスリンを静脈循環に一定の速度で注入する。血糖値の低下を一致させるには、D50が注入される。 D50注入速度は、取り込みの速度よりも大きい場合、グルコースの上昇が発生します。同様に、速度が全身のグルコース取り込みを一致させるには不十分な場合、ドロップが発生します。安定した血糖測定値が達成されるまでグルコースの滴定は継続。この安定した期間中の血糖値およびグルコース注入率を記録し、報告されている。結果は、全身のインスリン感受性のインデックスを提供しています。技術は特定の実験的な要件を満たすように改良できます。それは組織特異的インスリン刺激によるグルコース取り込みだけでなく、全身のブドウ糖の代謝回転を決定することができる放射性トレーサーの使用によってさらに強化されています。

Watch this Scientific Journal Video about Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat at JoVE.com

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

高インスリン血症、正常血糖クランプは、インスリン作用の評価のための&quot;ゴールドスタンダード&quot;です。インスリンはグルコース取り込みを刺激する一定の速度で注入する。このドロップダウンに対抗するために注入された外因性グルコースの量は、インスリン感受性の指標である。ここで手順は意識、無拘束のラットで実行されます。

覚醒ラットにおける高インスリン血症、正常血糖クランプ

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

hyperinsulinemic-euglycemic-clamp-in-the-conscious-rat

Nanyang Technological University

Research

JoVE Journal

Medicine

51.9K ビュー.  University of Calgary. 高インスリン血症、正常血糖クランプは、インスリン作用の評価のための"ゴールドスタンダード"です。インスリンはグルコース取り込みを刺激する一定の速度で注入する。このドロップダウンに対抗するために注入された外因性グルコースの量は、インスリン感受性の指標である。ここで手順は意識、無拘束のラットで実行されます。

ビデオ: Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

意識、無拘束マウスにおける高インスリン血症、正常血糖クランプ

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

医学

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

虚血ラットの肺の血管新生

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

ラット網膜へのエルバックス40Wを通じて徐放性ドラッグデリバリー：慢性症状の治療のための含意

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

配慮ラットモデルにおける血行動態の措置、煎茶や血液サンプリングを実行するために頸動脈と頸静脈のカテーテル

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

コンシャスげっ歯類における腎機能の経皮評価

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

ラットにおけるジメチルニトロソアミン誘発性肝線維症モデル

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

ラット肝臓肝門部クランプモデルを用いたダイレクトセグメント別肝臓内送達方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

再門脈部分肝移植のラット体内血管指向 70% 肝切除

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

ダブル室容積脈波による意識下マウスにおける呼吸機能の評価

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

意識的に拘束されたウサギにおける発作、不整脈および無呼吸の識別のためのマルチシステムモニタリング

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...