Name: visualization and quantification of brown and beige adipose tissues in mice using [18f]fdg micro-pet/mr imaging
Uploaded: 2021-07-01T14:00:00
Description: Watch this Scientific Journal Video about Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging at JoVE.com

我們感謝中國國家自然科學基金會（NSFC）-優秀青年科學家基金（香港及澳門）（81922079）、香港研究資助基金會一般研究基金（GRF 17121520 and 17123419）及香港研究資助基金會合作研究基金（CRF C7018-14E）對小動物影像實驗的支持。

下文所述的规程遵循香港大学的动物护理指引。研究中使用的动物是8周龄的C57BL / 6J小鼠。
1. 动物外科手术和感冒挑战
<ol><li>进行肩胛间 BAT （iBAT） 解剖。<ol>
<li>通过腹膜内注射氯胺酮/木拉嗪（100mg / kg体重氯胺酮和10mg / kg体重木拉嗪）麻醉小鼠。麻醉后，将小鼠的毛发从颈部剃到肩胛骨正下方。</li>
		<li>消毒后将小鼠放在加热垫上，并沿着小鼠的背侧中线做一个2厘米的切...

<ol>
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J., Shi, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+brown+fat+in+mice+with+18F-FDG+micro-PET/CT.">Functional imaging of brown fat in mice with 18F-FDG micro-PET/CT.</a> Journal of Visualized Experiments: JoVE. 69, (2012).</li><li>Greenwood, H. E., Nyitrai, Z., Mocsai, G., Hobor, S., Witney, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+PET/CT+imaging+using+a+multiple-mouse+imaging+system.">High-throughput PET/CT imaging using a multiple-mouse imaging system.</a> Journal of Nuclear Medicine: Official Publication, Scoiety of Nuclear Medicine. 61 (2), 292-297 (2020).</li><li>Carter, L. M., Henry, K. E., Platzman, A., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printable+platform+for+high-throughput+small-animal+imaging.">3D-printable platform for high-throughput small-animal imaging.</a> Journal of Nuclear Medicine: Official Publication, Scoiety of Nuclear Medicine. 61 (11), 1691-1692 (2020).</li><li>Jal Andersson, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+cold-induced+brown+adipose+tissue+glucose+uptake+rate+measured+by+(18)F-FDG+PET+using+infrared+thermography+and+water-fat+separated+MRI.">Estimating the cold-induced brown adipose tissue glucose uptake rate measured by (18)F-FDG PET using infrared thermography and water-fat separated MRI.</a> Scientific Reports. 9 (18), 12358 (2019).</li><li>Eal Lundstrom, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+estimated+with+the+magnetic+resonance+imaging+fat+fraction+is+associated+with+glucose+metabolism+in+adolescents.">Brown adipose tissue estimated with the magnetic resonance imaging fat fraction is associated with glucose metabolism in adolescents.</a> Pediatric Obesity. 14 (9), (2019).</li><li>Eal Lundstrom, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+cooling-reheating+protocol+indicates+decreased+fat+fraction+via+lipid+consumption+in+suspected+brown+adipose+tissue.">Magnetic resonance imaging cooling-reheating protocol indicates decreased fat fraction via lipid consumption in suspected brown adipose tissue.</a> PLoS One. 10 (4), 0126705 (2015).</li><li>Nakamura, Y., Yanagawa, Y., Morrison, S. F., Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullary+reticular+neurons+mediate+neuropeptide+Y-induced+metabolic+inhibition+and+mastication.">Medullary reticular neurons mediate neuropeptide Y-induced metabolic inhibition and mastication.</a> Cell Metabolism. 25 (2), 322-334 (2017).</li><li>Jal Fueger, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+animal+handling+on+the+results+of+18F-FDG+PET+studies+in+mice.">Impact of animal handling on the results of 18F-FDG PET studies in mice.</a> Journal of Nuclear Medicine: Official Publication, Scoiety of Nuclear Medicine. 47 (6), 999-1006 (2006).</li><li>Vines, D. C., Green, D. E., Kudo, G., Keller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mouse+tail-vein+injections+both+qualitatively+and+quantitatively+on+small-animal+PET+tail+scans.">Evaluation of mouse tail-vein injections both qualitatively and quantitatively on small-animal PET tail scans.</a> Journal of Nuclear Medicine Technology. 39 (4), 264-270 (2011).</li></ol>

在本研究中，描述了基于PET / MR的小动物功能性棕色和米色脂肪组织的成像和定量。该方法使用不可代谢的葡萄糖类似物[18F]FDG作为成像生物标志物，以便以非侵入性方式识别具有高葡萄糖需求的脂肪组织。MR提供良好的软组织对比度，可以更好地将脂肪脂肪组织与邻近的软组织和肌肉区分开来。当与PET结合使用时，由于高葡萄糖利用，这可以对活化的脂肪细胞进行成像和定量。这里概述?...

为了响应不断变化的营养需求，脂肪组织作为能量缓存，采用脂质储存或动员模式来满足身体的需求1。此外，脂肪组织在体温调节中也起着关键功能，通过称为非颤抖产热的过程，也称为兼性产热。这通常由棕色脂肪组织（BAT）实现，其表达丰富的线粒体膜蛋白解偶联蛋白1（UCP1）。作为质子载体，UCP1 通过解耦质子传输和 ATP 产生来产生热量2。在冷刺激下，BAT中的产热通过激活交感神经系统（SNS）启动，然后释放去甲肾上腺素（NE）。NE与β3肾上腺素能受体结合，导致细胞内环AMP（cAMP）升高。因此，CREB（cAMP反应元件结合蛋白）的cAMP / PKA依赖性参与通过直接结合CREB反应元件（CRE）2刺激Ucp1转录。除BAT外，白色脂肪组织中还发现了棕色样脂肪细胞，因此被命名为米色或brite（白色棕色）细胞1，3。为了响应特定的刺激（如寒冷），这些原本静止的米色细胞被重塑以表现出多种棕色样特征，包括多位点脂滴，密集的线粒体和增强的UCP1表达3，4，5。
动物研究表明，棕色和米色脂肪细胞除了具有减脂作用外，还具有多种代谢益处，包括胰岛素致敏，降脂，抗炎和抗动脉粥样硬化6，7。在人类中，米色/棕色脂肪的含量与年龄、胰岛素抵抗指数和心脏代谢紊乱呈负相关8。此外，通过冷驯化或β3肾上腺素能受体激动剂激活人体米色/棕色脂肪细胞可防止一系列代谢紊乱4，9，10。这些证据共同表明，诱导棕色和米色脂肪组织是管理肥胖及其相关医学并发症的潜在治疗策略8。
有趣的是，尽管它们具有相似的功能，但米色和经典的棕色脂肪细胞来自不同的前体，并通过重叠但不同的机制激活1。因此，棕色和米色脂肪细胞的 体内 成像和定量对于更好地了解这些脂肪组织的分子控制至关重要。目前 ，18F-氟脱氧葡萄糖（[18F]FDG）正电子发射断层扫描（PET）扫描与计算机断层扫描（CT）相结合仍然是临床研究中表征产热棕色和米色细胞的金标准8。磁共振成像（MRI）使用强大的磁场和射频脉冲来产生详细的解剖结构。与CT扫描相比，MRI以更高的分辨率生成器官和软组织的图像。这里提供的是适应冷暴露后小鼠模型中功能性棕色和米色脂肪的可视化和定量的方案，这是诱导脂肪褐变的常见和最可靠的方法。该方法可用于高精度表征小动物模型中的产热脂肪库。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sterile saline</td>
			<td>BBraun</td>
			<td></td>
			<td>0.9% sodium chloride intravenous infusion, 500 mL</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Terumo</td>
			<td>SS05L</td>
			<td>5 mL syringe Luer Lock</td>
		</tr>
		<tr>
			<td>Dose Calibrator</td>
			<td>Biodex</td>
			<td></td>
			<td>Atomlab 500</td>
		</tr>
		<tr>
			<td>Eye lubricant</td>
			<td>Alcon Duratears</td>
			<td></td>
			<td>Sterile ocular lubricant ointment, 3.5 g</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Terumo</td>
			<td>10ME2913</td>
			<td>1 mL insulin syringe with needle</td>
		</tr>
		<tr>
			<td>InterView Fusion software</td>
			<td>Mediso</td>
			<td>Version 3.03</td>
			<td>Post-processing and image analysis software</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Chanelle Pharma</td>
			<td></td>
			<td>Iso-Vet, inhalation anesthetic, 250 mL</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Alfasan International B.V.</td>
			<td>HK-37715</td>
			<td>Ketamine 10% injection solution, 10 mL</td>
		</tr>
		<tr>
			<td>Medical oxygen</td>
			<td>Linde HKO</td>
			<td>101-HR</td>
			<td>compressed gas, 99.5% purity</td>
		</tr>
		<tr>
			<td>Metacam</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>5 mg/mL Meloxicam solution for injection for dogs and cats, 10 mL</td>
		</tr>
		<tr>
			<td>nanoScan PET/MR Scanner</td>
			<td>Mediso</td>
			<td></td>
			<td>3 Tesla MR</td>
		</tr>
		<tr>
			<td>Nucline nanoScan software</td>
			<td>Mediso</td>
			<td>Version 3.0</td>
			<td>Scanner operating software</td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Reflex 7</td>
			<td>203-100</td>
			<td>7mm Stainless steel wound clips, 20 clips</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alfasan International B.V.</td>
			<td>HK-56179</td>
			<td>Xylazine 2% injection solution, 30 mL</td>
		</tr>
	</tbody>
</table>

visualization and quantification of brown and beige adipose tissues in mice using [18f]fdg micro-pet/mr imaging

棕色和米色脂肪细胞现在被认为是肥胖和代谢综合征的潜在治疗靶点。非侵入性分子成像方法对于提供对这些产热脂肪库的关键见解至关重要。在这里，该协议提出了一种基于PET / MR成像的方法，以评估小鼠肩胛间棕色脂肪组织（iBAT）和腹股沟皮下白色脂肪组织（iWAT）中棕色和米色脂肪细胞的活性。当与MR成像提供的精确解剖信息相结合时，使用[18F]FDG（不可代谢的葡萄糖类似物）作为放射性示踪剂，实现了产热脂肪库的可视化和定量。在冷适应后7 d进行PET/MR成像，并在不同脂肪库中定量[18F]FDG信号，以评估产热脂肪组织的相对动员。去除iBAT大大增加了小鼠iWAT中的冷诱发[18F]FDG摄取。

在这项研究中，三组小鼠（每组n = 3）接受了微PET / MR成像，其中它们被安置在热中性（30°C）或冷（6°C）下7天。一组小鼠（n = 3）在冷处理前将其iBAT（iBATx）移除（图1A）。该方法导致所有三只小鼠中白色脂肪组织活性的改变。特别是，使用微PET/MR成像在iWAT中观察到[18F]FDG摄取显着增加（图1B-C）。这种共同配准的成像数据被证明?...

Watch this Scientific Journal Video about Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging at JoVE.com

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

使用基于微PET / MR成像的方法对小鼠中的产热脂肪库进行功能成像和定量。

使用[18F]FDG微PET / MR成像对小鼠中的棕色和米色脂肪组织进行可视化和定量

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

PET / MR成像允许对活体动物中这些代谢活性的棕色和米色脂肪细胞进行定性和定量测量，以便可以在功能基础上测量这些所谓的产热脂肪细胞的活性。该技术的主要优点是它允许在同一受试者上采集和组合PET和MRI扫描，从而能够精确和同时采集小动物不同库中的产热脂肪细胞。该方法可以很容易地转移到任何临床前系统中，或者通过同时对多只小鼠进行成像来修改为高通量格式，从而以更低的成本和时间增加成像数据的统计能力。为了收获肩胛间棕色脂肪组织，将麻醉的8周龄C57 Black 6小鼠放在加热垫上，并沿着背中线做一个2厘米的切口。从肩胛间区域取出iBAT垫。出血停止后，使用7毫米不锈钢缠绕夹关闭切口。收集完所有iBAT后，将小鼠安置在重症监护室14天。向小鼠施用每公斤5毫克美洛昔康六天，并在伤口愈合后立即取下夹子。要在成像会话之前启用自动顺序PET/MR扫描，请将判别级别设置为400至600千电子伏，置信模式设置为1-3，扫描时间为20分钟，研究同位素设置为F-18，以设置PET采集的工作流程。要获取 T1 加权磁共振以进行衰减校正，请设置梯度回波-3D 参数。要获取 T2 加权磁共振作为解剖参考，请设置快速自旋 Echo 2D 参数。要允许重建 PET 图像，请使用 Tera-Tomo 3D 算法，将重合模式设置为 1-3，并设置衰减、死区时间、随机、衰减和散射校正，以创建总体素大小为 0.3 立方毫米的图像。在成像研究开始前一天检查PET定量的准确性，穿上适当的PPE，并按照制造商的建议用F18-FDG填充5毫升注射器。使用剂量校准器记录注射器的活性并记录测量时间。在软件中，使用插值椭圆ROI在注射器的重建图像上绘制感兴趣的体积，并将恢复的活性与使用剂量校准器测量的值进行比较。在成像实验当天，使用镊子小心地将18F-FDG储备小瓶放在L-块桌面屏蔽罩后面，并将等分试样的放射性同位素稀释在100至150微升无菌盐水中，总活性浓度为200至250微居里。将18F-FDG溶液吸入装有针头的1毫升注射器中，并使用剂量校准器测量放射性，如图所示。在将18F-FDG溶液注入尾静脉之前，记录要成像的小鼠的重量。注意注射时间和注射器的残余放射性，以便进行衰变校正。然后将鼠标放回笼子中60分钟。要计算注射的18F-FDG活性，请从注射后测量的活性中减去注射前注射器中的活性。在放射性同位素摄取期结束时，打开微型PET/ MR扫描仪的鼠标床的空气加热器。将麻醉的FDG注射小鼠放在扫描仪床上的呼吸垫上，并执行侦察视图以确定鼠标位置。调整鼠标床的位置，使整个身体可以被观察，确保MRI视野的中心与身体的中心重合。在“PET 采集”下，选择“上一次采集的扫描范围”以使用侦察视图位置，然后单击“准备”将动物床从 MR 移动到 PET。选择“确定”以启动 PET 扫描。在放射性药物编辑器中记录18F-FDG给药前后测量的注射剂量和时间，并在“受试者信息”菜单下输入鼠标的重量。PET扫描完成后，选择准备移动到MR成像并在研究列表窗口中完成所有MR采集。选择“确定”以启动 MR 扫描。完成整个工作流程后，在后处理软件中简要评估采集的MR图像的质量，然后单击“主页”将鼠标床从MR移动到原始位置。对所有小鼠进行成像后，要重建原始扫描菜单中的数据，请选择PET采集以加载已完成的PET扫描，然后选择T1加权MR采集以允许创建材料图谱。然后使用Tera-Tomo 3D算法重建数据，如图所示。在冷处理之前去除iBAT会改变iWAT的活性，正如通过微PET / MR成像观察到iWAT中18F-FDG摄取显着增加所表明的那样。此外，在米色脂肪组织中观察到的多斑点脂肪细胞，与在iWAT中观察到的假手术动物的脂肪细胞形态相比，在iBAT去除后小鼠的iWAT中更明显。冷处理，假手术小鼠在iBAT中显示出显着升高的18F-FDG摄取。在冷处理前去除iBAT的小鼠在iWAT中显示出最高的18F-FDG摄取量。事实上，冷暴露导致假手术小鼠iBAT中18F-FDG摄取增加七倍，而冷诱导小鼠中iBAT的去除导致18F-FDG摄取增加八倍，而热中性小鼠仅观察到适度的两倍增加。当尝试这一过程时，将每只小鼠置于每个PET / MRI采集的相同位置对于改善不同区域中棕色和米色脂肪细胞的可视化和定量非常重要。使用这种方法并与其他MRI方法一起，例如通过成像或测温术进行扩散，可以开始更深入地了解小鼠中棕色和米色脂肪细胞的空间分布和患病率，这可以潜在地转化为人类。这种方法使研究人员能够研究这些产热的棕色和米色脂肪细胞的功能。因此，当研究非临床产热途径时，它们特别强大，其中这些经典标志物没有被改变或上调。

Research

JoVE Journal

Biology

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

科研

生物学

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

初白和棕色预生细胞与新生小鼠的隔离和分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

使用氧化氘定量测定棕色脂肪组织中 从头 脂肪酸合成

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration (Video) | JoVE 

基于脱细胞的骨骼肌脂肪浸润定量

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Assessment of Acinetobacter Biofilm Formation and Characterization of Biofilms

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization (Video) | JoVE

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...