この研究は、国立衛生研究所(NIH)、助成金R00-DK114498、および米国農務省(USDA)、助成金CRIS:3092-51000-062 Y.Z.によってサポートされています。

動物は、ベイラー医科大学(BCM)の施設動物ケアと使用委員会によって承認された動物ケアと実験プロトコルに従って標準化された条件で収容されています。動物は標準的または特別な食事、水のアドリビタムを与えられ、12時間の昼/夜のサイクルで保たれます。
1. 断食血清脂質の測定
<ol><li>マウスを午後5時以降に新しいケージに移し、水への自由なアクセスで速く、一晩(実験前に約16時間の断食で)一晩の断食は、マウスの胃腸管の完全な空を保証します。 注:マウスは断食中に便を食べるので、食物の撤退は十分に断食されていることを保証できません。</li>
	<li>翌朝、尻尾でマウスをつかみ、表面に置きます。マウスの尻尾を軽く引き戻し、マウスをリカーナに配置します。マウスを呼吸させながらマウスの動きを再訓練するために、鼻の拘束を置きます。鼻の拘束のつまみを締めます。胸の動きを監視して、動物が正常に呼吸していることを確認し、マウスが拘束者に費やす時間を最小限に抑えます。</li>
	<li>遊離マウスの尾静脈に表面的な切開(ニック)を作り、切開から25μLの血液を、マウスを拘束することなくガラス毛細血管(毛細管の約1/3を充填)に引き込みます。マイクロ遠心チューブに素早く血液を吹き込みます。</li>
	<li>スタイプティックパウダーを使用して出血を止め、ケージ内の飼料を補充し、マウスにストレスの兆候がないことを確認します。</li>
	<li>すべてのマウスの血液離脱を完了します。</li>
	<li>室温で1時間妨げなままにして血液を凝固させる。冷蔵されたベンチトップマイクロ遠心分離機で4°Cで2,000 x gの凝固した血液サンプルを10分間回転させ、上清(血清)を分析に移します。 注:血清は、分析まで数週間–20 °Cで保存することができます。長期保存の場合は、血清を-70°Cに保ちます。</li>
	<li>メーカーが提供するプロトコルを使用して、各脂質代謝物を分析します。</li>
</ol>2. 経口脂質耐性試験
<ol><li>午後5時以降、翌日に与えられる脂質内容積の計算のためにマウスを秤量する。次に、マウスを新しいケージに移し、一晩(16時間)速くします。</li>
	<li>翌朝、1つのケージに収容されたマウスの尾をマークし、その後の出血ステップでそれらを識別するのに役立ちます。</li>
	<li>尾静脈にニックを作り、切開から15μLの血液をガラス毛細血管に引き込み(毛細管の約1/5を充填)、T = 0血清のマイクロ遠心チューブに素早く血液を吹き込みます。 注:マウスに過剰な出血がない限り、アッセイ中に出血を止める必要はありません。</li>
	<li>ガバジマウスは、18Gガビンギ針を1グラム当たり15μlの割合で使用し、前断食体重を用いた20%の脂質内である。1分で各マウスをスタックします。 注:動物の重量を量り、適切なギャバジ針のサイズを決定します。一般的に、18ゲージのガバジ針はマウス>25gに適しており、20〜22ゲージのガバジ針はより小さな動物に適しているであろう。マウスを擦り付け、肩の上の皮膚をつかんで動物の頭を所定の位置に保持します。口の中に針を入れ、食道に達するまで、上口蓋に沿って穏やかに進みます。チューブは抵抗なしで通過する必要があります。適切な深さに達すると、脂質内をゆっくりと投与することができる。イントラリピッドを管理した後、針の挿入時に同じ角度に従って針を静かに取り除きます。ケージに動物を戻し、呼吸や他の苦痛の兆候を監視します。 注:経口ギャバジや尾出血の技術で経験の浅い研究者は、2分以上の時間で各マウスを積み重ねることができます。</li>
	<li>T= 1、2、3、4、5、および6時間で血液を引き出す:尾出血を介してマウス1個あたり15μLの血液(1/5毛細血管)を引き出し、すぐにマイクロ遠心管に血液を吹き込みます。</li>
	<li>マイクロ遠心分離機で10分間室温で2,000 x gで血液サンプルを回転させます。浮遊脂肪層を含む上清を、貯蔵用のPCRチューブに移します。上清は、分析まで数週間–20°Cで保存することができます。 注:上清はプラズマでなければなりません。一部のサンプルが遠心分離の時点で既に凝固している場合、トリグリセリド測定には影響しません。</li>
	<li>最後の採血後、スタイプティックパウダーを使って出血を止め、ケージ内の飼料を補充し、マウスが極端なストレスの兆候を示さないことを確認します。</li>
	<li>トリグリセリド標準の2μLをロードし、96ウェルプレートに上清を収集しました。</li>
	<li>トリグリセリド試薬を200μL加え、37°Cでプレートを5分間インキュベートして色を現します。</li>
	<li>実験室の版の読者の参照波長の500 nmの吸光度を測定し、サンプルの濃度を計算する。</li>
</ol>3. β3 アドレナリン受容体アゴニスト CL 316,243 刺激リポリシスアッセイ
<ol><li>CL 316,243を無菌生理食物で5mg/mL(50x)のストック溶液として調製し、使用するまで-20°Cで保存します。</li>
	<li>午前中、マウスの重量を量って、実験に必要な希釈CL 316,243溶液の量を計算する。マウスは、1 mg/kg体重の最終投与のために、希釈されたCL 316,243の体重1グラム当たり10 μLを受け取ります。</li>
	<li>マウスを水への自由なアクセスを持つ新しいケージに移し、4時間速くします。</li>
	<li>生理液を使用して50xストックから十分な1x CL 316,243ソリューションを作ります。1x CL 316,243溶液の最終濃度は0.1 mg/mLです。対照処置群には生理的な処置を使用する。</li>
	<li>出血のステップの間に容易に識別するために同じケージに収容されたマウスの尾をマークします。</li>
	<li>尾静脈にニックを作り、切開から15μLの血液をガラス毛細血管(毛細管の約1/5を充填)に引き込み、T = 0サンプルのマイクロ遠心チューブに素早く血液を吹き込みます。 注:マウスに過剰な出血がない限り、アッセイ中に出血を止める必要はありません。</li>
	<li>希釈したCL 316,243溶液(または実験に含まれる場合は制御)を10μL/g体重の体積で腹腔内に注入します。1分で各マウスをスタックします。60分間の実験ごとに最大5匹のマウス、または2人のチームには10匹のマウスを使用します。</li>
	<li>T= 5、15、30、60分で血液を引き出す:尾出血を介してマウスあたり15μLの血液(1/5毛細血管)を引き出す。</li>
	<li>最後の採血後、スタイプティックパウダーを使って出血を止め、ケージ内の飼料を補充し、マウスが極端なストレスの兆候を示さないことを確認します。</li>
	<li>冷蔵マイクロ遠心分離機で5分間、4°Cで2,000 x gで血液サンプルを回す。上清をPCRチューブに移して保存します。上清は分析まで数週間–20°Cで貯えることができる。</li>
	<li>2x連続希釈グリセロール標準(0.156、0.312、0.625、1.25、および2.5 mg/mlトリオレーヌ相当濃度)の1μLをロードし、96ウェルプレートに上清を集めます。100 μLのフリーグリセロール試薬を加え、37°Cでプレートを5分間インキュベートさせて発色させます。</li>
	<li>実験室用プレートリーダーを用いて540nmで吸光度を測定し、試料の濃度を計算します。</li>
</ol>

<ol>
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著者らは開示するものは何もない。

3つのアッセイは、いくつかの重要な考慮事項を持つラボで堅牢に機能を記述しました。一晩断食は、断食血清脂質レベルおよび経口脂質耐性試験を決定するために必要とされる。経口脂質耐性試験では、特に1時間と2時間の時点で、脂肪層の形成を最小限に抑えるために室温で血液を回す必要があります。この脂肪層が形成された場合、この脂肪層を捨てないようにすることが重要です。脂?...

炭水化物と脂質は、エネルギー代謝の 2 つの主要な基質です。.異常な脂質代謝は、II型糖尿病、心血管疾患、脂肪肝疾患、癌を含む多くのヒト疾患をもたらす。食事脂質は、主にトリグリセリドが腸内を通ってリンパ系に吸収され、心臓近くのクロミクロンの静脈循環に入る1。脂質は血流中のリポタンパク質粒子によって運ばれ、そこで脂肪酸部分は筋肉および脂肪組織2などの末梢器官におけるリポタンパク質リパーゼの作用によって遊離される。残りのコレステロールが豊富な残骸粒子は肝臓3によってクリアされる。マウスは、脂質代謝を研究する研究モデルとして研究室で広く使用されています。包括的な遺伝的ツールセットが利用可能で、比較的短い繁殖サイクルで、脂質がどのように吸収され、合成され、代謝されるのかを研究するための強力なモデルです。
脂質代謝の複雑さのために、高度なリピドミクス研究または同位体トレーサー研究は、通常、脂質種または脂質関連代謝フラックスおよび運命のコレクションを定量化するために使用される。これは、特殊な機器や専門知識を持たない研究者にとって大きな課題を生み出します。本論文では、技術的に挑戦的な技術を用いる前に、初期テストとして役立つ3つのアッセイを紹介する。これらはマウスの非末端手順であり、脂質処理能力の潜在的な違いを特定し、影響を受けるプロセスを絞り込むのに非常に有用です。
まず、断食血清脂質分子を測定することは、マウスの全体的な脂質プロファイルを確認するのに役立ちます。マウスは、食後に多くの脂質種が上昇し、増加の程度が食事の組成によって強く影響を受けるので、断食する必要があります。総コレステロール、トリグリセリド、非エステル化脂肪酸(NEFA)を含む多くの脂質分子は、吸光度を読み取ることができる市販キットやプレートリーダーを用いて測定することができる。
第二に、脂質耐性の経口検査は、脂質処理能力を吸収および代謝の正味の効果として評価する。経口投与された脂質は、トリグリセリド濃度の循環(1~2時間)のスパイクを引き起こし、その後、血清中性脂肪レベルは基底レベル(4〜6時間)に戻る。このアッセイは、マウスが外因性脂質をどの程度うまく処理できるかに関する情報を提供します。心臓、肝臓、および褐色脂肪組織はトリグリセリドの活性消費者であり、白い脂肪組織はそれをエネルギーリザーブとして保存する。これらの関数の変更は、テスト結果の違いにつながります。
最後に, 保存された脂質を動員する脂肪分解を促進することは、減量のための可能な戦略と考えられています。.脂肪組織におけるβ3-アドレナリン受容体シグナル伝達経路は脂肪細胞脂肪分解において重要な役割を果たし、ヒト遺伝学は肥満と相関するβ3アドレナリン受容体におけるTrp64Argの機能喪失多型Trp64Argを同定した。CL 316,243は、特異的かつ強力なβ3-アドレナリン受容体アゴニストで、脂肪組織脂肪分解およびグリセロールの放出を刺激する。CL 316,243に対するマウスの反応の評価は、化合物の有効性の開発、改善、および理解に関する貴重な情報を提供することができる。
総称して、これらの試験は、マウスの脂質代謝状態の変化の初期画面として使用することができる。それらは器械および試薬のアクセス性のために選ばれる。これらのアッセイから得られた結果により、研究者は動物の代謝適合性の全体像を形成し、より洗練されたターゲットを絞ったアプローチを決定することができます。

<table>
	<tbody>
		<tr>
			<td>20% Intralipid</td>
			<td>Sigma Aldrich</td>
			<td>I141</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes 1ml</td>
			<td>Shaotong</td>
			<td>B07F1KRMYN</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316,243 Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5976</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Feeding Needles (18 Gauge)</td>
			<td>Kent Scientific</td>
			<td>FNC-18-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco</td>
			<td>50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Plus Chemistry Reference Kit</td>
			<td>Verichem</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-222-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematrocrit Capillary Tube, Not Heparanized</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td></td>
		</tr>
		<tr>
			<td>NEFA STANDARD SOLUTION</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Triglycerides Reagent</td>
			<td>Fisher Scientific</td>
			<td>TR22421</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagents</td>
			<td>Thermo Scientifi</td>
			<td>TR13421</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>11695-4022-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing whole-body lipid-handling capacity in mice

脂質代謝の評価は代謝機能の基礎であり、生体内代謝研究に不可欠と考えられています。脂質は、その合成と代謝に関与する多くの経路を持つ多くの異なる分子のクラスです。.栄養と肥満の研究のための脂質止まりの評価の出発点が必要です。本論文では、習得にほとんど専門知識や実践を必要とし、マウスの脂質代謝異常をスクリーニングするためにほとんどの研究室で適応させることができる3つの簡単でアクセス可能な方法について説明します。これらの方法は、(1)経口脂質耐性試験を通じた食事脂質取扱能力に対する栄養学的脂質処理能力に対する(2)の市販キットを用いたいくつかの断食血清脂質分子を測定し、(3)医薬化合物CL316,243、マウスにおける反応を評価する。これらの方法を合わせて、マウスにおける脂質処理能力の概要を提供する。

我々は、各アッセイがマウスの脂質代謝に関する貴重な情報を提供することを3つの抜粋で示す。C57BL/6J雄マウスは、8週齢から8週間の高脂肪食(HFD)摂食に挑戦し、総コレステロール値が有意に上昇したのに対し、血清トリグリセライドとNEFAは有意に上昇しなかった(表1)、血液中のトリグリセリドおよびNEFAは主に食物脂肪チャレンジによって調節されないことを示唆している。マ?...

Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

Assessing Whole-Body Lipid-Handling Capacity in Mice

本論文では、マウスの脂質代謝を評価するための3つの簡進性の高いアッセイを提供する。

マウスにおける全身脂質処理能力の評価

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

assessing-whole-body-lipid-handling-capacity-in-mice

Research

JoVE Journal

Medicine

3.8K ビュー.  Baylor College of Medicine. 本論文では、マウスの脂質代謝を評価するための3つの簡進性の高いアッセイを提供する。

ビデオ: Assessing Whole-Body Lipid-Handling Capacity in Mice

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

マウスにおける経胸壁心エコー

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

マウスの長い持続時間の全身温熱療法のためのプロトコル

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局所または全身薬理学的介入後のマウスの揮発性全身麻酔の感度変化を評価する

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

マウスにおける運動耐容能および心臓適応に関する化学的に誘発卵巣障害の影響を研究するための方法

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

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 ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

抗がん剤の特異性を評価します<em&gt;インビトロ</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

リン-31磁気共鳴分光法：測定するためのツール<em&gt;インビボ</emヒト骨格筋における&gt;ミトコンドリア酸化的リン酸化能力

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

人間の活動の地域の定量化と全身褐色脂肪組織18F-FDG PET/CT を使用

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

ヒンドリム虚血のマウスモデルにおける治療血管新生の評価

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...