这项工作得到国家卫生研究院（NIH）的支持，授予R00-DK114498，美国农业部（美国农业部）向Y.Z提供3092-51000-062。

根据贝勒医学院（BCM）机构动物护理和使用委员会批准的动物护理和实验协议，动物被安置在标准化条件下。动物被喂食标准或特殊的饮食，水广告脂质，并保持12小时的日夜循环。
1. 测量禁食血清脂
<ol><li>下午5点以后将老鼠转移到一个新的笼子里，并快速免费取水，过夜（实验前大约16个小时的禁食）。通宵禁食确保完全排空小鼠的胃肠道。 注意:老鼠在禁食时吃粪便，所以食物提取不能确保它们被充分禁食。</li>
	<li>第二天早上，抓住老鼠的尾巴，把它放在水面上。轻轻地拉回鼠标尾部，将鼠标放在抑制器中。将鼻子约束重新训练鼠标的运动，同时仍然允许鼠标呼吸。拧紧鼻子约束的旋钮。监控胸部运动，确保动物正常呼吸，并尽量减少鼠标在抑制器中停留的时间。</li>
	<li>在自由移动的鼠标的尾部静脉中做一个表面切口（尼克），从切口中抽出25μL的血液，制成玻璃毛细血管（填充大约1/3的毛细血管），而不抑制鼠标。迅速将血液吹入微中心管中。</li>
	<li>使用型粉停止出血，在笼子里补充饲料，并确保小鼠没有出现压力的迹象。</li>
	<li>完成所有小鼠的血液抽取。</li>
	<li>让血液凝固，使其在室温下不受干扰1小时。在冷藏台式微中心中，在 4 °C 下以 2，000 x g 旋转凝固的血液样本 10 分钟，并转移超高血清（血清）进行分析。 注:血清可在 +20 °C 下存储数周，直到分析。对于长期存储，将血清保持在 +70°C。</li>
	<li>使用制造商提供的协议分析每个脂质代谢物。</li>
</ol>2. 口服脂内耐受性测试
<ol><li>下午5点以后，称量小鼠，计算第二天交给它们的脂内体积。然后，将老鼠转移到一个新的笼子里，并禁食它们过夜（16小时）。</li>
	<li>第二天早上，将老鼠的尾巴放在一个笼子里，以帮助识别它们在随后的出血步骤中。</li>
	<li>在尾部静脉上划一个刻痕，从切口中抽出 15 μL 的血液到玻璃毛细血管中（填充大约 1/5 的毛细血管），并快速将血液吹入 T = 0 血清的微中心管中。 注意:除非小鼠出现过度出血，否则无需在检测过程中止血。</li>
	<li>Gavage 小鼠使用 18G 盖瓦奇针的 20% 内脂，每克体重为 15 μl，使用禁食前体重。将每只鼠标堆叠 1 分钟。 注:称量动物，确定适当的咽针大小。一般来说，18号的咽针适合25克>小鼠，20-22号的咽针更适合较小的动物。擦伤老鼠，抓住肩膀上的皮肤，把动物的头抱到位。将咽针放入口腔，然后沿着上味轻轻推进，直到食道到达。管子应该通过没有阻力。一旦达到适当的深度，内脂可以慢慢管理。在插入针头时，在管理脂内后，轻轻取出相同角度的针头。将动物送回笼子，并监测有呼吸困难或其他不适的迹象。 注:缺乏口腔咽部或尾部出血技术经验的研究人员可以将每只小鼠堆叠2分钟甚至更长时间。</li>
	<li>在 T = 1、2、3、4、5 和 6 小时抽血:通过尾部出血每只小鼠抽取 15 μL 的血液（1/5 毛细管），并迅速将血液吹入微中心管中。</li>
	<li>在室温下以 2，000 x g 旋转血液样本，在微中心中旋转 10 分钟。将超高纳特（包括浮动脂肪层）转移到 PCR 管中存储。超高纳特可以在 +20 °C 下存储数周，直到分析。 注:超高等离子体应该是等离子体。如果某些样品在离心时已经凝固，则不会影响甘油三酯测量。</li>
	<li>最后一次采血后，停止使用型粉出血，在笼子里补充饲料，并确保小鼠没有表现出极度紧张的迹象。</li>
	<li>装载 2 μL 甘油三酯标准，并将超分子收集到 96 井板中。</li>
	<li>加入 200 μL 甘油三酯试剂，让板在 37 °C 下孵育 5 分钟，用于颜色开发。</li>
	<li>测量实验室板读卡器中 500 nm 的吸血量，参考波长为 660 nm，并计算样本的浓度。</li>
</ol>3. β3 肾上腺受体激动器 CL 316，243 刺激利波利西斯检测
<ol><li>准备 CL 316，243 作为无菌盐水中 5 毫克/mL （50 倍） 的库存溶液，并储存在 ±20°C，直到使用。</li>
	<li>在早晨，称量小鼠，计算实验所需的稀释CL 316，243溶液量。小鼠将得到10μL每克体重稀释CL 316，243，为1毫克/千克体重的最后剂量。</li>
	<li>将老鼠转移到一个新的笼子里，免费取水，并禁食4小时。</li>
	<li>使用盐水从 50 倍库存中制作足够的 1x CL 316，243 溶液。1x CL 316，243 溶液的最终浓度为 0.1 毫克/mL。使用盐水进行对照组。</li>
	<li>标记存放在同一笼子里的老鼠的尾巴，以便在出血步骤中轻松识别。</li>
	<li>在尾部静脉中划出一个刻痕，从切口中抽取 15 μL 的血液到玻璃毛细血管中（填充大约 1/5 的毛细血管），并快速将血液吹入微中心管中，用于 T = 0 样本。 注意:除非小鼠出现过度出血，否则无需在检测过程中止血。</li>
	<li>以 10 μL/g 体重的体量，在腹地注射稀释的 CL 316，243 溶液（或如果包含在实验中）。将每只鼠标堆叠 1 分钟。每60分钟的实验最多使用5只小鼠，或为双人小组使用10只小鼠。</li>
	<li>在 T = 5、15、30、60 分钟抽血:通过尾部出血每只小鼠抽取 15 微升血液（1/5 毛细血管）。</li>
	<li>最后一次采血后，停止使用型粉出血，在笼子里补充饲料，并确保小鼠没有表现出极度紧张的迹象。</li>
	<li>在冷藏微中心中，在 4 °C 下以 2，000 x g 旋转血液样本 5 分钟。将超纳特转移到 PCR 管进行存储。超高纳特可以在 +20°C 下存储数周，直到分析。</li>
	<li>负载 1 μL 的 2 倍串序稀释甘油标准 （0.156， 0.312， 0.625， 1.25 和 2.5 毫克/毫升三聚氨酯当量浓度）， 并收集超分子到 96 井板.加入100微升免费甘油试剂，让板在37°C孵育5分钟，使颜色形成。</li>
	<li>使用实验室板读卡器测量 540 nm 的吸血量，并计算样品的浓度。</li>
</ol>

<ol>
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作者没有什么可透露的。

这三个分析描述了实验室中强有力的功能，并考虑了几个关键问题。夜间禁食是确定禁食血清脂质水平和口腔脂内耐受性测试所必需的。对于口腔脂内耐受性测试，在室温下旋转血液以尽量减少脂肪层的形成至关重要，尤其是在 1 小时和 2 小时的时间点:重要的是不要丢弃这个脂肪层，如果它形成。确保将超高肽与脂质层一起转移，并轻轻地将它们混合在一起，以进行甘油三酯测定。
<p class="jov...

碳水化合物和脂质是能量代谢的两大基质。异常脂质代谢导致许多人类疾病，包括II型糖尿病、心血管疾病、脂肪肝疾病和癌症。膳食脂质，主要是甘油三酯，通过肠道吸收到淋巴系统，并进入心脏1附近的血小龙静脉循环。脂质由血液中的脂蛋白颗粒携带，脂肪酸脂肪通过脂蛋白脂酶在肌肉和脂肪组织2等外周器官的作用而释放。剩余的富含胆固醇的残留颗粒由肝脏3清除。老鼠在实验室中被广泛用作研究脂质代谢的研究模型。拥有全面的遗传工具集和相对较短的繁殖周期，它们是研究脂质如何被吸收、合成和代谢的有力模型。
由于脂质代谢的复杂性，复杂的脂质学研究或同位素示踪研究通常用于量化脂质物种或脂质相关代谢通量和命运的集合4，5。这给没有专门设备或专业知识的研究人员带来了巨大的挑战。在本文中，我们提出了三个测试，在使用具有技术挑战性的技术之前，这些测试可以作为初始测试。它们是小鼠的非终端程序，因此非常有助于识别脂质处理能力的潜在差异和缩小受影响的过程。
首先，测量禁食血清脂质分子可以帮助确定小鼠的整体脂质特征。老鼠应该禁食，因为许多脂质物种在饭后会上升，而且增加的程度受到饮食成分的强烈影响。许多脂质分子，包括总胆固醇、甘油三酯和非酯化脂肪酸（NEFA），都可以使用商业套件和可读取吸血的板读器进行测量。
其次，口服脂质耐受性测试将脂质处理能力评估为吸收和代谢的净效应。口服的脂内导致循环甘油三酯水平激增（1至2小时），之后血清甘油三酯水平恢复到基底水平（4至6小时）。此检测提供了有关鼠标处理外源性脂质的程度的信息。心脏、肝脏和棕色脂肪组织是甘油三酯的活跃消费者，而白色脂肪组织将其储存为能量储备。这些功能的变化将导致测试结果的差异。
最后，促进脂溶解以调动储存的脂质被认为是减肥的可能策略。脂肪组织中的β3-肾神经受体信号通路在脂肪细胞脂解中起着重要作用，人类遗传学已经识别出与肥胖相关的3-肾上腺受体中功能丧失的多态性Trp64Arg。CL 316，243，一种特异且有效的β-肾上腺素受体激动剂，刺激脂肪组织脂解和甘油释放。评估鼠标对CL 316，243的反应可以提供关于化合物的开发、改进和理解的宝贵信息。
总的来说，这些测试可以用作小鼠脂质代谢状态变化的初始屏幕。它们是为仪器和试剂的可访问性而选择的。通过这些检测结果，研究人员可以形成动物代谢适应的整体图景，并决定更复杂和更有针对性的方法。

<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

评估脂质代谢是评估代谢功能的基石，它被认为是活体代谢研究的关键。脂质是许多不同的分子的一类，其合成和代谢涉及许多通路。需要一个起点来评估营养和肥胖研究的脂质血脂。本文描述了三种简单易行的方法，这些方法几乎不需要专业知识或实践来掌握，并且大多数实验室都可以将其改编为筛查小鼠的脂质代谢异常。这些方法是（1）测量几个禁食血清脂质分子使用商业套件（2）通过口服脂质耐受性测试，以评估饮食脂质处理能力，（3）评估对药物化合物CL 316，243的反应，在小鼠。这些方法将共同提供小鼠脂质处理能力的高水平概述。

我们用三段摘录显示，每次检测都提供了有关小鼠脂质代谢的宝贵信息。对于C57BL/6J雄性小鼠来说，从8周大开始接受8周高脂肪饮食（HFD）喂养的挑战，总胆固醇水平显著升高，而血清甘油三酯和NEFA则没有（表1），这表明血液中的甘油三酯和NEFA不是主要受膳食脂肪挑战的调节。在第二组小鼠中，C57BL/6J和C57BL6/N子系统从8周大开始喂养HFD8周。他们的血清甘油三酯水平在口腔脂内挑战后?...

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

本文为评估小鼠脂质代谢提供了三种简单易行的检测。

评估小鼠全身脂质处理能力

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 次观看.  Baylor College of Medicine. 本文为评估小鼠脂质代谢提供了三种简单易行的检测。

视频: 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.

经胸超声心动图在小鼠

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

科研

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.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

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.

持续时间较长的小鼠全身热疗协议

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.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

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.

一个方法研究的化学诱导卵巢早衰的运动能力和适应心脏的影响在小鼠

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.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

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.

评估抗癌药物的特异性<em&gt;体外</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.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

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.

18F-fdg Petct 对活性人棕色脂肪组织的全身和区域定量

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;

评估下肢缺血的Murine模型的治疗血管生成

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