著者は、DHMRI CLAS動物ケアスタッフの強い支持を認めるファシリティマネージャー氏ダニエル・ペラルタと獣医師、博士Glicerioイグナシオ、DVM MRCVSに出席したいと思います。

全ての動物手順は、プロトコル14から017の下で、ノースカロライナ州リサーチキャンパス（NCRC）に位置デビッドH.マードック研究所IACUCによって承認された、糖尿病および糖尿病前症/インスリン抵抗性と治療の有効性の非ヒト霊長類モデルの特徴は、改善するために、インスリン感受性および代謝機能。 1.動物の選択および研究の準備<ol><li>毎月の食物摂取および体重の記録に基づいて選択食事および体重安定動物。 注：彼らの食物摂取量が安定するまで食欲の最近の下落を示した動物を特徴とすることがないようにしてください。 <ol><li>成熟した動物のために（&gt; 5から6年）、その体重ヶ月連続の間に最初のサルを検査し、体重の劇的な変化のための代謝状態を変更する以外の原因を除外することなく、10％を超えて異なる動物を選択しないでください。 </li></ol></li><li>グルコース、インスリンおよびc-peptiのためデ検体は、プロテアーゼ阻害剤（アプロチニンとDPP4I）とK 2 EDTAサンプル採取チューブを準備します。 注：DPP4I +アプロチニンカクテルは、プロテアーゼ阻害の広いスペクトルを提供し、これは追加の検体（グルカゴン、GLP-1）のために必要とされるべきです。追加の検体が収集されない場合には、血液チューブはサンプル収集の一貫性を維持するために、同じ方法で調製されるべきです。この方法のために検証されていないアッセイ用の試料は、製造業者の推奨に従って、個別に収集する必要があります。 <ol><li> DPP4I 10mlのアプロチニン凍結乾燥100mgのを混合することにより、プロテアーゼ阻害剤カクテルを準備します。採取した血液のあらゆるミリリットルのために各血液チューブにアプロチニン+ DPP4I液10μlを追加します。 </li><li>可能なコレクション超過のために各チューブにプロテアーゼ阻害剤カクテルの追加の10μlのを追加します。使用するまで-20℃で処理した血管を保管してください。 。手順の間に濡れた氷の上にチューブを保ちます再、4℃でスピン。 </li><li>標準血清化学分析のためのベースラインでの血液サンプルを収集するために血清分離管を使用します。全血球数（CBC）のために、プロテアーゼ阻害剤なしで標準K 2 EDTAを使用します。血液サンプルをスピンダウンされた後に一定分量の血漿および血清に凍結バイアルを使用してください。 </li><li>動物の識別、日付、手順、時点、およびサンプル量で適切に血管と凍結バイアルにラベルを付けます。適切なアッセイのための血漿中の分析物（単数または複数）とクリオバイアルにラベルを付けます。 </li></ol></li><li>通常の生理食塩水の250ミリリットル袋にmlのヘパリン1,000 USP単位の0.15ミリリットルを注入することにより、ヘパリン化生理食塩水洗浄を準備します。 0.06 mgのヘパリン/ミリリットルの溶液を得ました。サンプル間のフラッシングのために生理食塩水ロックへのこの溶液60ミリリットル - 40を描画します。それぞれ、注入前と後のブドウ糖注入ポートをフラッシュするために別々のシリンジ中に追加の1ミリリットルと5ミリリットルを描画します。 </li></ol> 2。動物の鎮静と準備<ol><li>動物のケージから手順の前下回らない14時間、およびせいぜい18時間の食品を削除します。 注：これは、動物は血糖値のいずれかの食後の変動を回避するための手順のために絶食することが重要です。また、麻酔をかけながら、逆流、胃内容物の吸引を避けるために予防措置です。 </li><li> 10mg / kgの、一般的な麻酔薬として筋肉内投与ケタミンとIVGTT手順の期間中、落ち着いた動物。処置中に、必要に応じて30分、または - 20の間隔で - （10mg / kgの5）追加のケタミンを投与します。 <ol><li>鎮静させた動物を計量。加熱された手順テーブル上の横方向臥位で動物を置きます。 </li><li>動物が麻酔の安定した平面内にあることを確認するための臨床パラメータごとに15〜20分を監視します。パルスオキシメータと- （200 BPM 100）およびSPO 2（&gt; 92％）、心拍数を測定します。呼吸数を測定します（20から50呼吸/分）のw<html>ストップウォッチi番目、15秒かけて視覚的に、または手で呼吸を数えると4を掛けます。測定温度（&gt; 97°F）直腸に。ガムと唇（しっとり、ピンク）の周りの粘膜の色を監視します。 </li></ol></li><li> 2カニューレサイトを準備します。カテーテルが挿入される関心領域から毛をトリム、および交互クロルヘキシジンのスクラブと70％アルコールで領域全体を滅菌するためにバリカンを使用してください。 <ol><li>左または右橈側または伏在静脈の領域で1つのカテーテルを配置し、三方活栓でヘパリン生理食塩水洗浄（0.06 mgのヘパリン/ミリリットル）に取り付けます。これは、サンプリング採血サイトです。 </li><li>頭部または伏在静脈の領域における別の脚や腕に第二のカテーテルを配置し、ポートを接続します。ブドウ糖注入のためにこのサイトを使用してください。前ブドウ糖注入カニューレ特許を維持するために、小さなヘパリン添加生理食塩水の1ミリリットルフラッシュを使用してください。 </li></ol></li></ol>e_title &quot;&gt; 3。IVGTT手順注：IVGTT手順は8採血サンプリング時点（ 表1）で構成されています。 <ol><li>ベースラインのサンプルを取り、空腹時血糖値を測定するために手持ちグルコメーターを使用しています。動物の全体的な健康状態を評価するために、CBCのための標準的な化学分析のために血清サンプルだけでなく、全血サンプルを入手します。製造元の指示8、9通りにマカクで使用するための検証キットを使用して、ベースラインサンプルからのグルコースおよびインスリンレベルをアッセイするために血漿サンプルを収集します。 注：前のカニューレのデッドスペースに残留血液やヘパリンを除去するために、血液採取のための任意のサンプルを採取するカニューレから採取した0.5ミリリットルのプレドローを有することが重要です。 </li><li>ベースラインサンプルを得た後、ブドウ糖注入ポートに30秒以上、50％デキストロース（250ミリグラム/ kg）の用量を注入。 注記：高用量モデル（500ミリグラム/ kg）を使用することができ、用量は、長手方向の比較を行うための手順を横切って固定しなければならないのに。 <ol><li>ポートに残されたブドウ糖がないことを確認するために、ヘパリン添加生理食塩水5mlで注入ポートをフラッシュします。注入の終了はT0です。注入からの残留ブドウ糖がその後の血液サンプルを汚染する可能性があるとして、技術者が、手袋を交換してください。 </li></ol></li><li>最初の注入後サンプル時間点はT5分、T7分、T10分、T15分、T20分、最後のサンプル時点はT30の分であるが続くブドウ糖点滴の終わりから、T3分です。 （ステップ3.1参照）、ベースラインサンプルとアッセイグルコースおよびインスリンレベルに各時点から血漿を収集します。 <ol><li>時点分T3で、再び血糖値をチェックするために手持ちグルコメーターを使用しています。 注：ベースライン時血糖計の測定値とT3は、ブドウ糖の点滴を確認するだけです。時点T3分で血糖値は、約100ミリグラム/ dlの高い共同でなければなりません空腹時ベースライン血漿グルコースレベルにmpared。 </li></ol></li></ol> 4.動物の回復とサンプル処理<ol><li>カニューレを取り外し、時点分のT30後の止血のためにカテーテルを挿入部位に圧力を加えます。それは意識を取り戻したと座ってするまで動物を監視します。動物一度オファー供給が完全に回復されます。 </li><li>直ちに氷上にK 2 EDTAチューブに各全血試料を配置します。コレクションの10分以内に4℃の温度で3000 rpmで遠心分離します。分析まで-80℃で凍結バイアル、凍結ストアにアリコートの血漿サンプル。 <ol><li>標準血清化学分析のために血清チューブに血液が下回らない20分、室温で3,000 rpmで遠心分離する前に、せいぜい30分間室温で静置します。コレクションの48時間以内にアッセイするまで血清サンプルをフリーズします。 注：としてまでCBC分析のために収集冷蔵全血サンプルコレクションの24時間以内にサイード。 </li></ol></li></ol> 5.データ処理<ol><li>血漿インスリン及びグルコース曲線を確立した後、ベースライン16、17以上のグルコース値の自然対数の傾きからのグルコースクリアランス率Kを決定します。 注：健康な動物は、多くの場合、30分以内にベースライングルコース値に戻るように健康なNHPは、2またはより多くの場合大きく、ウェル1上のグルコースクリアランス率Kを有することが期待されてもよいです。インスリン産生がオフに低下すると、グルコースクリアランス率Kが1を下回る、より劇的に低下します。 </li><li> T30 16、17を介して、時点間の各線分の曲線下面積を表す台形の面積の合計の和として、全体としてのAUCを計算します。 注：伝統的に、手順の最初の10分のAUCは、acutと考えられています手順の最後の20分のAUCが遅くインスリン応答（LIR）と考えている間にインスリン反応eは、（AIR）をグルコースへ。動物はより多くの代謝障害になると、インスリンAUCは、インスリン非感受性を増加させるための補償を反映して、増加します。顕性糖尿病への動物に遷移すると、しかし、AUCは、AIRによって捕獲、急性インスリン期にしばしば最初は、減少します。 </li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+biochemical+changes+in+insulin+dependent+and+non-insulin+dependent+diabetes+mellitus+and+their+role+in+the+development+of+secondary+complications.">Serum biochemical changes in insulin dependent and non-insulin dependent diabetes mellitus and their role in the development of secondary complications.</a> Int J Diab Dev Countries. 17, 33-37 (1997).</li><li>Shirasaki, Y., Yoshioka, N., Kanazawa, K., Maekawa, T., Horikawa, T., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+physical+restraint+on+glucose+tolerance+in+cynomolgus+monkeys.">Effect of physical restraint on glucose tolerance in cynomolgus monkeys.</a> J Med Primatol. 42, 165-168 (2013).</li></ol>

著者は、代謝性疾患の分野で積極的な開発業務受託機関（クラウンバイオサイエンス）と提携しています。

IVGTTアッセイから、空腹時血糖およびインスリンレベルが達成される。体重5、12、13に基づいて単一のブドウ糖注入によるグルコース刺激インスリン放出の能力を評価し、それは動物の能力を評価することを可能にしますインスリンを放出し、ベースラインに向かって上昇したグルコースレベルを返します。これは正常なグルコースおよびインスリンレベル健常対照、正常血糖と高イ...

IVGTT日常異なる代謝状態5,7にヒトにおけるβ細胞機能を決定するために使用される便利な機能的アッセイである。T2Dの動物モデルでは、それは井戸から代謝性疾患の進行を示した動物を特徴づけるためのツールとして認識されています代謝異常、高血糖状態8、9に健康。T2Dの最も近い動物モデルは、アカゲザルとカニクイザルが顕著な例であるのヒト以外の霊長類（NHPsを）、で実証されています。これらの動物は自然に年齢や肥満の同じ危険因子は、ヒト10のようにその発生率に貢献してT2Dを開発しています。代謝異常疾患が11を進行するにつれてさらに、同様の疾患の進行およびアミロイド沈着を示す膵臓の病状があります。 ここでは、これらの動物における代謝状態の私達のコロニーの特性評価の一部としてNHPsをでIVGTTを実行する当社の標準的な方法について報告します。この方法は、かかる他の、より多くの時間とコストのかかる技術2に対して実行するのは簡単。 IVGTTは、迅速かつ頻繁に動物の大きなコロニーを特徴付けるために有用です。糖化ヘモグロビン（HbA1C値）、動物の食事と食物摂取の歴史、ならびにそれらのパーセント除脂肪体重と体脂肪のレベルを考慮に入れた場合、IVGTTは、顕性糖尿病6に向けて、動物の代謝状態および進行を特徴づけるため、通常は十分です、8。 HbA1C値は、3ヶ月前の6週間のグルコースレベルの信頼できる測定を提供する、赤血球の寿命の平均血糖レベルを表します。 IVGTTの絶食ベースライン血液サンプルから測定した場合、この値は、手順の間に数ヶ月の間に血糖コントロールにウィンドウを提供します。彼らの最後のIVGTT、その前の値よりもはるかに高いのHbA1c値が示すことになるので、動物は代謝異常から糖尿病に移行した場合その前の値に近い方のHbA1c値は、彼らはごく最近に移行したことを示すであろう、一方の移行は、彼らの最後のIVGTT後すぐに始まったこと。一般的には、アカゲザルでは、HbA1C値が6％より大きい値異常と考えられ、血糖コントロール不良10、23を示しています。 血糖レベルは、全体として、動物の行動や一般的な健康の文脈の中で解釈されるべきです。糖尿病マカク - 展示過食症、多飲、および多尿 - 人間のように。動物のグループハウジングは、これらの指標の測定と代謝異常や糖尿病のサルのために必要な個々のケアに重要な課題を提供します。我々は単独より個人ケアを提供することができるようにするために、動物を収容するお勧めし、より容易にサルの健康行動マーカー8を監視します。さらに、糖尿病のマカクは増加した（体重減少、ならびに上昇した脂質プロフィールを示すであろうコレステロール、高トリグリセリド血症）および血清化学の乱れミネラル代謝。それをこれらの臓器への損傷は、しばしば代謝障害/糖尿病を進めるの合併症であるとして、血清化学で肝臓と腎臓機能のマーカーを測定することが重要であり、血糖、脂質、ミネラル不均衡9の同時決定することができ、11、18、24 。 この方法を使用する場合は、サルの寿命を介して複数の、頻繁な特徴付けから生成された歴史的な値は特に価値があります。このようなグルコースクランプまたは段階的なグルコース注入（GGI）などの他の手順は、完全に動物の健康状態を評価するために必要とされている場合は、その履歴が利用できない場合、それは初期特性時に一般的です。ベースラインが確立された後、しかし、3ヶ月ごとの周波数の繰り返しIVGTTsは、動物の進行状況を追跡するのに通常は十分です。動物は全体の複数の研究に登録されている場合、このことは特に重要です暦年では、それらの代謝状態に基づきます。自分の健康は、一度に年間は比較的安定したままかもしれないが、動物の代謝状態が悪化したときに、インスリン抵抗性と耐糖能異常の劇的な増加は、非常に急速に発生する可能性があります。 HbA1c値は、3ヶ月間隔でスケジュールされた手順の間、動物の健康状態の低下や改善のいくつかの補間を可能にします。この理由から、この方法は、それらの天然の寿命に亘って複数、縦研究に使用される動物を特徴づけるために理想的です。

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characterization of metabolic status in nonhuman primates with the intravenous glucose tolerance test

静脈内グルコース負荷試験（IVGTT）は、グルコース恒常性の特性評価において重要な役割を果たしています。摂食及び絶食状態の両方における血中グルコースレベルを含む血清生化学的プロフィール、HbA1c値、インスリンレベル、食事、身体組成、体重状態の病歴と一緒になった場合に、正常および異常な血糖コントロールの評価を行うことができます。 IVGTTの解釈はブドウ糖挑戦に関連して時間をかけてグルコースおよびインスリンレベルの変化の測定を介して行われます。考慮すべき重要なコンポーネントは次のとおりです：T0（グルコース注入の終わり）、最初の20分で急速なグルコースクリアランスの勾配由来のグルコースクリアランス率K（T20へのT1）、時間に関連して到達したピークグルコースおよびインスリンレベルグルコースベースライン、および曲線下面積（AUC）に戻ります。これらのIVGTT対策は健康トンからグルコース恒常性の移動に伴って特徴的な変化を表示しますOA病気の代謝状態5。ここで我々は、肥満代謝障害に、ヒトにおけるII型糖尿病（T2D）の最も関連性の高い動物モデルとIVGTTとリーン健康からこれらの動物の臨床プロファイルであるヒト以外の霊長類（アカゲザルとカニクイザルマカク）、の特徴を説明します、そして、T2D状態8、10、11。

図1に示した結果は、30分のIVGTTの過程で、成熟した健康と糖尿病のカニクイザルからの典型的なグルコースおよびインスリン曲線の実証されています。健康的で先進的な糖尿病のサルからのデータは、代謝特性評価の範囲の両方の両極端からの動物の間で明らかな違いを対比するために示されています。このIVGTTプロトコルは、同様の結果とアカゲザルで...

Watch this Scientific Journal Video about Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test at JoVE.com

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

このプロトコルの目的は、非ヒト霊長類において血糖コントロールを評価し、健康の代謝異常にそれらの代謝状態を評価するために静脈内グルコース負荷試験（IVGTTs）を実行するための標準的な方法を提示することです。

静脈内グルコース負荷試験とヒト以外の霊長類における代謝状態のキャラクタリゼーション

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

characterization-metabolic-status-nonhuman-primates-with-intravenous

Research

JoVE Journal

Medicine

10.8K ビュー.  Crown Bioscience. このプロトコルの目的は、非ヒト霊長類において血糖コントロールを評価し、健康の代謝異常にそれらの代謝状態を評価するために静脈内グルコース負荷試験（IVGTTs）を実行するための標準的な方法を提示することです。 

ビデオ: Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAPのテストバッテリー：リスニングの意味を理解する

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

医学

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

マウスの脳室内（ICV）と静脈（IV）注入による治療薬のデリバリー

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance

Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1.
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7.
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11 or sublingual nitrate12,13. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14, with a high positive response rate immediately after administration15. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1.
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1&plusmn;0.6 min16,17. This allows not only diagnosis and pathophysiological assessment19-22, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.

We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.

コンバインド下半身陰圧とチルト試験：起立耐性を測定するための &quot;ゴールドスタンダード&quot;

Orthostatic tolerance (OT)  refers to the ability to maintain cardiovascular stability when upright,  against the hydrostatic effects of gravity, and ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

高齢者の短期漸進的筋力トレーニングによる筋力・筋力・筋弛緩力の改善

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

マウスの膵臓の組織学的解析と代謝プロファイリングに対する郭清

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

マウスの脳内へ連続的な薬剤注入システムと末梢グルコース不耐症に視床下部のインスリン シグナルを勉強

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

経口ブドウ糖負荷試験 (OGTT) とインスリン負荷試験 (ITT) を使用して高脂肪食飼育マウスの生体内糖代謝の研究

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

ドキソルビシンの球後注射と徐放性アプロチニンによるボリューム保持の予防によるマウスのネフローゼ症候群の誘導

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

免震を用いた消化管ホルモン分泌のメカニズム潅流ラット小腸

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

非アルコール性脂肪性肝炎 (NASH) の解像度を評価するためにマウスで存続可能肝生検の手順の定款

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...