作者希望感谢志愿者参与这些研究, 以及波士顿医疗中心的外科工作人员提供脂肪组织活检。Dr. Gokce 由国立卫生研究院 (NIH) 资助 HL081587、HL114675 和 HL126141。Dr. Farb 由 NIH 资助 K23 HL135394。

此处所述的协议和示例是由波士顿大学医学院机构审查委员会 (IRB, 议定书 #H-25644) 批准的, 并按照《赫尔辛基宣言》进行。所有受试者在参与前均提供书面知情同意. 
 1. 溶液的制备和微玻璃套管 <ol> <li> 准备 4-2-hydrosyethyl-1-piperazineethanesulfonic 酸性盐水 (HEPES) 溶液。1升, 溶解8.059 克氯化钠, 0.298 克氯化钾, 0.296 g MgSO 4 和 #8226; 7H 2 O、0.235 g CaCl 2 和 #8226; 2H 2 o、0.16 g、 2 PO 4 、0.01 克 EDTA、1.081 克 d-葡萄糖和2.383 克 HEPES 酸950毫升的去离子水.用去离子水把容积设置为1升。用氢氧化钠调节 pH 值7.4。HEPES 缓冲区可存储在4和 #176 的7天; C. </li>
	<li> 准备20x 盐缓冲库存。1升, 增加143.8 克氯化钠, 7.0 克氯化钾, 5.9 g MgSO 4 , 和 7.4 g CaCl 2 和 #8226; 2H 2 O 进入900毫升蒸馏水。用去离子水来弥补1升的体积。存储库存解决方案在4和 #176; C. </li>
	<li> 准备20x 缓冲库存。1升, 增加26.8 克 NaHCO 3 和0.2 克 EDTA 和 #903; 2H 2 O 成900毫升的去离子水。用去离子水来弥补1升的体积。存储库存解决方案在4和 #176; C. </li>
	<li> 准备在生理学实验中使用的克雷布斯解决方案。为1升, 添加900毫升去离子水, 50 毫升的20x 缓冲库存, 50 毫升的20x 盐缓冲液, 0.99 克 d-葡萄糖和 0.16 g 的 2 PO 4 。使用 HCl 将 pH 值调整到7.4。保持缓冲液的 pH 值, 不断搅拌, 连续地盖上石蜡或气泡. 
	<ol> <li> 检查每20分钟的 pH 值, 使克雷布斯解决方案每天都新鲜. </li>
	</ol> </li> <li> 使微玻璃套管的内径为 40-240 和 #181; m 使用商业可用的针/吸管拉拔器。玻璃套管的大小取决于被隔绝的血管的内径大小 (50-350 和 #181; m)。另外, 购买预定尺寸的微玻璃套管. </li>
</ol> 2。试剂的制备 <ol> <li> 准备乙酰胆碱, 10 -2 M, 库存解决方案)。在10毫升去离子水中溶解18.29 克。存储1毫升等分的库存解决方案 (10 -2 M) 在-80 和 #176; C。在实验的天连续地稀释获得以下工作的集中:10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 m </li>
	<li> 准备罂粟碱 (2 x 10 -2 M, 库存解决方案)。在10毫升去离子水中溶解0.07517 克。准备10和 #181; 等分库存解决方案, 存储在-80 和 #176; c. 串行稀释获得 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 M 在实验的当天. </li>
	<li> 准备 endothelin-1 (ET-1, 2 x 10 -5 M, 库存解决方案)。溶解50和 #181; g endothelin-1 在1毫升 1% BSA/PBS。存储1毫升等分的库存解决方案 (2 x 10 -5 M) 在-80 和 #176; C。在实验当天稀释以获得 10 -10 M 的工作浓度. </li>
	<li> 在-20 和 #176 中存储试剂; c 或高达-80 和 #176; c 取决于可用的冰箱类型, 但不长于6月. </li>
</ol> 3。脂肪组织收集和血管制备 <ol> <li> 招募肥胖男性和女性 (体重指数 (BMI) 和 #8805; 35 公斤/米和 #178; 年龄和 #8805; 18 年) 在波士顿医疗中心 (BMC) 参加减肥手术计划。共有40受试者参加了这些实验. </li>
	<li> 在执行手术的外科医生计划的减肥手术中收集皮下和内脏脂肪组织样本. 
	<ol> <li> 分别从下腹壁和大网膜的内脏脂肪中获取皮下脂肪组织。典型的脂肪组织样本大小不同, 从 3-10 毫克的组织. </li>
		<li> 将组织立即放置在冷 HEPES 缓冲液中, pH 值为 7.4, 存储在4和 #176; C 为 24 h. 
		注: 此外, 脂肪动脉可以从精益个人以及其他类型的手术, 如疝气修补或整形手术, 也可以从皮下仓库活检通过跨皮腹脂肪垫活检类 = "xref" > 6 . </li>
	</ol> </li> <li> 使用组织解剖显微镜、微剪刀和微钳, 小心地从小脂肪动脉 (50-350 和 #181; m 腔内直径, 2-3 毫米) 清除周围的脂肪和结缔组织. </li>
	<li> 在插管玻璃毛细管管上使用细小的尼龙或丝线缝合动脉上的所有分支。仔细解剖的小动脉, 因为即使是轻微的破坏动脉墙造成重大功能变化. 
	<ol> <li> 最小化容器暴露于光和热, 因为它可能导致血管舒张。由于有时很难区分小动脉和静脉, 通过咬住血管的尖端来识别血管的大小和平滑肌张力。小动脉通常较小, 表现出更大的色调与静脉. </li>
	</ol> </li> <li> 准备风琴室。用10毫升注射器慢慢地用克雷布斯溶液填充玻璃毛细管管和橡胶管. </li>
	<li> 一旦橡胶管被填满, 玻璃毛细管管被浸没在克雷布斯溶液内, cannulate 在安全的管上安全的微动脉, 并小心地将动脉的两端与尼龙或丝缝合结绑在一起. </li>
	<li> 将克雷布斯溶液填满2毫升的风琴室. </li>
	<li> 用倒置显微镜 (放大倍数 10 x/0.25 目标) 和摄像机将风琴室固定到舞台上. </li>
	<li> 打开在 real-time 中以1赫 (1 帧/秒) 采样速率传输的边缘检测软件. </li>
	<li> 将加压油管的一侧连接到克雷布斯溶液填充的无微气泡的压力库, 因为气泡可以引入实验误差。将加压油管的另一侧连接到风琴室. </li>
	<li> 将克雷布斯溶液填充压力贮器连接到带清洁油管的压力传感器. </li>
	<li> 通过调节两个水库的高度来控制 intra-luminal 流; 因此, 当水库处于同一高度时, 不会发生 intra-luminal 流. </li>
	<li> 所有这些过程完成后, 打开加热块和软件程序, 以保持37和 #176 的温度; c. 连续灌注克雷布斯溶液和通气的容器, 气体混合物为 5% CO 2 , 21% O 2 和 74% N 2 7 , 8 贯穿整个实验过程. </li>
	<li> 实现所需的压力 insi分离的容器的流明逐渐增加腔内压力 (5 柱, 每5分钟) 通过肌界面面板的压力控制单元, 这样做的速度慢, 以避免内皮层损伤. 
	注: 一旦压力达到60柱, 需要20-30 分钟的平衡周期来稳定容器。所有血管功能研究在60柱和37和 #176; C 在 pH 值7.4。 7 </li> </ol> 4。脂肪微血管功能的评价 注意: 一般情况下, 脂肪动脉内皮依赖性血管舒张可以被诱发, 以响应生理 (流诱导) 和药理刺激 (乙酰胆碱诱导). 
<ol> <li> 流介导的内皮依赖性舒张调节 <ol> 在加压后, 记录脂肪动脉在休息 (Di) 的直径。这被称为静止基线直径. </li>
		<li> 预压缩血管到55% 的休息基线直径 (Dp), 增加1和 #181; endothelin-1 (ET-1, 10 和 #8722; 10 M) 直接到浴缸, 等待5分钟的效果。重复此过程, 直到达到所需的〜 55% pre-constricted 状态. </li>
		<li> 继 pre-constriction 后, 在相同和相反的方向上, 使脂肪组织微动脉的腔内间隙持续流动, 从而在不改变平均内腔压力的情况下, 在容器中形成压力差60柱. 
		注: 例如, 如果一个油藏在高度上移动10厘米, 另一个水库需要向下移动10厘米, 以改变压力梯度, 从而改变腔内流速。如果需要更精确的流量测量, 可将定量流量计系统应用于实验装置. </li>
		<li> 测量流介导的膨胀 3-5 分钟后启动的流动诱导。腔内流 (压力梯度) 的水平可以在 0-100 cmh-的范围内 2 o. 增加每个压力梯度的增量 #916; 10 cmh- 2 O 每 5-6 min, 最多 100 cmh- 2 o. 
		注: 为了在腔内诱导稳定的层流, 微玻璃套管的尖端尺寸需要非常接近小动脉内径;否则, 它会产生腔内的湍流流, 并诱发不需要的测量误差. </li>
		<li> 在流量介导的膨胀度评估后, 返回压力油藏到同一高度 (60 柱). </li>
		<li> 然后, 立即将溶液从腔室中除去, 并用新鲜的克雷布斯溶液取代动脉和室。这样做是谨慎的, 以不扰乱会议厅内的暂停动脉. </li>
		<li> 重复此过程 3-4 次, 20-30 分钟, 或直到孤立的船只返回到静止的基线直径. </li>
	</ol> </li> <li> 乙酰胆碱介导的, 内皮依赖性, 血管舒张 <ol> 当脂肪动脉已回到休息基线直径, pre-constrict 船只〜55% 由管理 endothelin-1 (ET-1, 10 <li>和 #8722; 10 M) 直接到浴缸, 如上文4.1.2 节所述. </li>
		<li> 以下 pre-constriction, 依次管理增加剂量 (2 和 #181; L) 乙酰胆碱是受体介导的, 一氧化氮激动剂 (乙酰胆碱, 10 -9 到 10 -5 M) 直接到浴缸。记录的变化, 在动脉直径5分钟后, 每剂量的管理. </li>
		<li> 一旦在最后的乙酰胆碱剂量的管理下到达一个高原, 用克雷布斯溶液清洗容器 3-4 次。允许 20-30 分钟的船只恢复和返回到休息基线直径。为了帮助维持 pH 值在器官的弧度, 改变克雷布斯解决方案每15分钟. </li>
	</ol> </li> <li> 孵育动脉与 N w -硝基-l-精氨酸甲酯 (l 名称, 10 -4 M), 一氧化氮合酶抑制剂30分钟, 然后重复4.2.1 和 4.2.2, 以表征的相对贡献一氧化氮对乙酰胆碱介导的血管舒张. </li>
	<li> 通过对增加剂量罂粟碱 (10 -8 到 10 -4 M) 的连续管理来评估内皮依赖性血管平滑肌功能和血管的生存能力, 直接到浴缸。如果血管直径不返回, 或超过休息基线 (Di) 的反应罂粟碱 ( 即 适当的血管舒张), 考虑船只不和丢弃数据点. </li>
</ol> 5。数据分析和计算 <ol> <li> 计算血管的反应性。使用百分比血管舒张率的数据表达, 以考虑基线差异的船只直径和计算使用以下公式: % 血管舒张 = (dt dp/dp) 和 #215; 100 其中 DT 是记录的直径在给定的时间点 (最大直径), Dp 是在添加收缩剂 ( i. e 后记录的直径。ET-1 诱导收缩直径), 而 Di 是在加入收缩剂 (静止基线直径) 之前记录的直径。 9 </li> <li> 将血管舒张和收缩敏感性 (剂量-反应) 定义为激动剂的浓度为乙酰胆碱, 罂粟碱, 或 ET-1, 引出50% 的最大反应 (EC 50 ), 可以定义的sigmoidal 参数和绘制, 如前所述。 10 注意: 在我们的实验室中, 脂肪微血管舒张的观察再现性研究有一个高相关系数 (CC) 0.99 (n = 10 容器实验). </li> </ol>
 6. 统计分析 <ol> <li> 将连续测量表示为平均值和 #177; SEM。这些往往是正态分布的。重复测量 two-way 分析脂肪微动脉血管反应性. </li>
	<li> 另一种方法是, 将曲线下的区域与治疗组间的剂量反应进行比较, 以进行累计血管舒张。在所有情况下, 考虑 P 和 #160; 和 #60; 0.05 统计意义. </li>
</ol>

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作者没有披露或利益冲突的报告。

从周围组织中剥离和分离脂肪动脉可能是一个费时和 labor-intensive 的过程, 并仔细注意细节和技术协议。该显微解剖程序需要细致的技能和专门的解剖用具, 以防止对平滑肌或血管内皮细胞层的潜在损害。即使在动脉壁微小的意外穿刺也能防止腔内加压, 并导致不成功的实验。此外, 利用缝线的小动脉插管对确保血管末端的玻璃毛细管吸管尖端之间的微动脉是至关重要的。
人孤立微血管可以发展自发的肌张力后 30-40 分钟加压在60柱。然而, 我们发现, 人脂肪小动脉一般大小 50-350 µm 内腔直径是相对较小的血管平滑肌的稀疏层, 并倾向于不发展显着肌张力与骨骼肌动脉。因此, 我们诱导 pre-constriction 这些小动脉使用 ET-1 前接触血管。我们发现, ET-1 施加一个更可预测的收缩反应, 而在某些情况下, 可能有变化的反应, α受体激动剂, 如肾上腺素。必须选择适当的激动剂, 以诱导一个理想的〜 55% pre-constriction;然而, 选择正确的 pre-constricting 剂量, 以防止过量的剂量和对容器的毒性损害也是至关重要的。我们做到这一点, 从较低的范围剂量和高达滴定的效果, 如4.1.2 节所述。
脂肪组织中的动脉对 pH 值和温度的变化敏感;14因此, 在实验过程中监测和控制适当的 pH 值 (7.4) 和温度 (37 ° c) 是至关重要的。虽然 videomicroscopy 是一个严密调控的温度控制系统, 这是当务之急, 以监测 pH 的克雷布斯溶液在整个实验, 以避免转移, 可能扭曲的结果。连续起泡的克雷布斯溶液和更换的解决方案每15分钟加压和洗涤步骤将减少 pH 值变化随着时间的推移。
虽然 videomicroscopy 是一种有用的技术来检测微血管的功能性质, 该技术有一个很大的学习曲线, 可能是劳动密集型, 特别是在血管隔离过程中。根据活检的组织类型, 我们对脂肪组织微血管的经验表明, 插管程序可能需要大量的时间和耐心。此外, 有固有的时间敏感性的实验, 因为身体外的船舶往往失去其生理和功能的特性随着时间的推移。因此, 这些实验需要在手术活检后的几小时内, 在最后的组织衰变之前进行。
这个实验模型的临床相关性和基本原理, 利用 videomicroscopy 的研究动脉是支持我们的能力, 直接探查病理生理学在完整的整个部分的人血管从生活的主体,无法通过非侵入性成像复制。事实上, 我们的能力, 直接访问和检查功能失调的人的血管, 例如一个肥胖的身体, 可以提供我们的机会, 以了解的途径, 在疾病的差异改变的情况下, 发现小说治疗目标。此外, 从我们的实验室和其他人的公布数据表明,体内脂肪动脉功能的评估与体内系统的内皮功能反应相关, 在个体内的其他血管床中,心血管危险因素包括高血压、吸烟、糖尿病和炎症。12,15,16我们还发布了数据, 证明了与内脏脂肪内皮细胞一氧化氮相关的免疫组化结果与肱动脉流介导血管舒张的相关性脂肪和全身循环的平行异常。17鉴于内皮功能紊乱的系统性, 我们认为这项技术不仅是研究人体血管组织的一种实用方法, 而且还可能利用这种方法在动物和临床纵向研究中检查治疗效果。其他检查微血管的技术也可以使用, 如线 myography, 这往往是在技术上难以执行, 并允许测量的等距张力的孤立船只, 以各种激动剂利用力传感器。虽然这种方法也提供了有关微血管的生理特性的信息, 但这种设置并没有利用 intra-luminal 压力系统, 这可能使它更不容易重述在体内的条件。videomicroscopy 的优点是能够诱导一个相对恒定的腔内压力, 最大限度地减少管腔中切变的变化。该方法可用于人类和动物的类似大小微血管。9,12,18最后, 应该指出的是, 有许多商用的 videomicroscopy 系统可供购买和设置;然而整体生理概念是相当一致的横跨所有平台。2,6,18

Videomicroscopy 是一种有用的技术, 用于检查从活着的人的主体前体去除小动脉的血管功能。我们的实验室专注于解剖从不同的脂肪组织隔间微小的微血管, 以表征各种脂肪环境的影响。这项技术的一个主要优点是, 从人体中取出的血管仍然是功能性的, 在活检后几分钟到几小时内就可以很容易地检查。通过微玻璃套管在腔内间隙中模拟和稳定的壁压力, 从而重现许多体内特征。1,2此外, 使用自动边缘检测软件进行可靠的 videomicroscopy 设置, 可以对内皮依赖性和独立的血管扩张剂和收缩的分离能力进行定性和定量评估。血管在 real-time, 允许快速的生理评估反应的物理和药理刺激。3其他微血管技术也可以使用, 如导线 myography, 它的耗时更少, 并通过力传感器测量各种激动剂的张力反应。
我们的实验室已经应用 videomicroscopy 来研究肥胖和血管功能紊乱之间的关系, 重点研究不同脂肪组织领域对脉管的影响。中央肥胖与腹内内脏脂肪的积累一直是最密切联系的因子生产, 代谢功能紊乱, 和代谢风险。据推测, 脂肪组织功能障碍与生产过剩的 pro-atherogenic 细胞因子和脂肪失调是强烈牵连在这些过程中, 但具体的调控分子和治疗指标仍然很大程度上发现.4此外, 在局部脂肪组织水平, 毛细血管稀疏和受损灌注已与脂肪组织 pseudohypoxia 和代谢失调。心血管疾病可能是脂肪组织功能紊乱的结果的假说正在演变。从脂肪释放的亲动脉粥样硬化调解人, 特别是与中央/内脏肥胖的联系, 可能促进内皮功能障碍和致病血管的变化, 可以在当地的脂肪血管显着, 并检测使用本文介绍了方法学。5
孤立性脂肪组织动脉的功能评估是一个有用的方法, 以获得理解的病理生理学和分子机制, 导致血管功能障碍的人肥胖。为了研究有助于脂肪库特异性功能障碍的机制, 我们已经制定了检查血管内皮依赖性和独立血管反应的方法, 并评价了各种调节在减肥手术时从肥胖患者身上获得的内脏和皮下 (SC) 脂肪组织标本的候选者。

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Chemical Name</td>
			<td style="width:159px;"></td>
			<td style="width:132px;"></td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acetylcholine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">A6625</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride (CaCl2)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td>223506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">D-(+)-Glucose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">G5767</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Endothelin-1</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E7764</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetra acetic acid (EGTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E3889</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Ethylene diamine tetra acetic acid (EDTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E9884</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">HEPES</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">H3784</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:352px;">Nw-nito-L-arginine methyl ester hydrochloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">N5751</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">M7506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride (KCL)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3911</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Potassium phosphate (KH2PO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P5655</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Papaverine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3510</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Sodium bicarbonate (NaHCO3 )</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S6014</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium chloride (NaCl)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S7653</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Sodium phosphate monobasic monohydrate (NaH2Po4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:127px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Finescience tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td>Zeiss Achromat</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laboratory tubing</td>
			<td>Euro-Pharm</td>
			<td>250100306F999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle/pippette puller</td>
			<td>David kopf instruments</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic monofilament nylon suture</td>
			<td>Surgical specialties</td>
			<td>A7756N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Finescience tools</td>
			<td>150000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vessel Chamber</td>
			<td>DMT</td>
			<td>VAS v.2.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of human adipose tissue microvascular function using videomicroscopy

虽然肥胖与新陈代谢和心血管疾病的发展密切相关, 但对这些过程的机制却知之甚少。这是假设, 从脂肪组织释放的 pro-atherogenic 调解人, 特别是与中央/内脏肥胖的联系可能会促进病原体血管的变化, 在当地和系统, 并认为心血管疾病可能是脂肪组织功能障碍的后果继续发展。在这里, 我们描述了一个独特的 videomicroscopy 的方法, 包括分析的血管扩张和收缩反应的完整小动脉从活的人体脂肪库去除。Videomicroscopy 是用来检测孤立微血管的功能性质, 以响应药理或生理刺激使用的压力系统模拟在体内条件。该技术是一个有用的方法, 以获得理解的病理生理学和分子机制, 导致血管功能紊乱局部在脂肪组织环境。此外, 脂肪组织微血管的异常也与全身性疾病有关。我们运用这项技术来检查在肥胖人群中的特定于仓库的血管反应。我们评估了内皮依赖性血管舒张的增加流量和乙酰胆碱在脂肪动脉 (50-350 µm 内径, 2-3 毫米) 从两个不同的脂肪库从同一个人的减肥手术中分离出来。我们证明, 从内脏脂肪的动脉表现受损内皮依赖性血管舒张与从皮下仓库分离。研究结果表明, 内脏微环境与血管内皮功能障碍有关, 这可能与临床观察相关, 将内脏肥胖增加与系统性疾病机制联系起来。videomicroscopy 技术可用于检查不同脂肪库的血管表型, 并对不同程度肥胖和代谢功能障碍的个体进行比较研究。该方法还可用于对临床干预的反应纵向检查血管反应。

我们的实验室使用 videomicroscopy 检查内皮依赖性和独立血管舒张, 以及脂肪组织动脉的 vasocontractile 功能从皮下和内脏脂肪的肥胖的人。特性实验设置显示在图 1A中。脂肪组织动脉悬浮在两个玻璃毛细管管之间, 并固定在器官腔内的缝合处, 如图 1B所示。如上文5.1 节所述, 动脉的反应可以通过在基线 (图 2A) 上检查容器, 然后是 pre-constriction 与 ET-1 到基线直径的 55% (图 2B), 然后由激动剂引起血管内腔的舒张和松弛, 如图 2C所示。
我们和其他人已经观察到, 内皮依赖性血管舒张反应增加流量 (剪切应力)11和乙酰胆碱12是显着迟钝的内脏相比, 皮下脂肪动脉 (图 3A, 3B) 在人类肥胖。然而, 在对罂粟碱的反应中, 内皮独立的血管舒张在两个仓库之间没有差异性改变 (图 3C)。这些结果表明, 内脏领域的血管功能紊乱主要是由于内皮细胞的功能紊乱, 至少在疾病的早期阶段。这项技术允许系统分析完整的小动脉的血管扩张反应, 也可以应用到人类脉管其他易接近的区域。ex 体内系统可以使用多种药理学方法进行操作, 如对胰岛素的反应作为血管内皮胰岛素抵抗的指标6, 或对硝酸甘油进行剂量反应研究, 以检测血管平滑肌层功能。12生物方法, 如沉默 RNA 也可以被引入13 , 以潜在的发展机制框架, 以了解关键的调节器, 赋予血管功能障碍在特定疾病条件。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56079/56079fig1.jpg"> 
图 1: Videomicroscopy 设置.(A) 描述 videomicroscopy 器官室的组成部分的图像。(B) 人体脂肪动脉的图像安装在两个玻璃毛细管管之间的器官室。血管直径的变化响应生理和药理刺激, 可以检查和量化使用专门的分析软件和倒置相机附件在系统中。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56079/56079fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56079/56079fig2.jpg"> 
图 2: 使用压力 videomicroscopy 的微血管舒张的图示.(A) 休息基线动脉直径 (post-pressurization 直径, 没有激动剂或刺激: Di) (B) pre-constricted 直径 (ET-1 诱导收缩直径: Dp), (C) 激动性诱发的 vasodilated直径 (在乙酰胆碱之后增加了: DT)。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56079/56079fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56079/56079fig3.jpg"> 
图 3: 肥胖人皮下与内脏脂肪动脉的内皮依赖性和独立舒张功能.与皮下脂肪组织微动脉相比, 血管内皮依赖性舒张功能明显减弱 (A) 增加的流量 (配对皮下和内脏血管, n = 20), 或 (B)乙酰胆碱剂量反应与/没有 l-名称 (配对皮下和内脏血管, n = 16)。(C) 在用罂粟碱剂量反应 (配对皮下和内脏血管, n = 8) 评估内脏和皮下动脉时, 内皮独立血管舒张没有发生差异性改变。* 表示车厂间的组别差异;†Denotes 车厂-与单纯乙酰胆碱相比, 乙酰胆碱与 l-名称的具体区别;NS: 不重要。数据显示为 mean±扫描电镜, p & #60; 0.05。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56079/56079fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy at JoVE.com

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

应用 Videomicroscopy 评价人脂肪组织微血管功能

Videomicroscopy 系统用于检测孤立脂肪组织动脉的功能特性, 以响应生理和药理刺激。该技术可用于研究肥胖人群不同脂肪组织领域的微血管表型。

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

assessment-human-adipose-tissue-microvascular-function-using

Nanyang Technological University

Research

JoVE Journal

Medicine

7.4K 次观看.  Boston University School of Medicine. Videomicroscopy 系统用于检测孤立脂肪组织动脉的功能特性, 以响应生理和药理刺激。该技术可用于研究肥胖人群不同脂肪组织领域的微血管表型。

视频: Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

科研

医学

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

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

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

在鼠害自觉肾功能的评估皮

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

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

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 ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

以基于技术的日常功能技能为目标的计算机化功能技能评估和培训计划

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

使用附睾白色脂肪组织进行脂肪覆盖的胰岛移植

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...