Name: assessment of human adipose tissue microvascular function using videomicroscopy
Uploaded: 2017-09-29T13:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy at JoVE.com

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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+murine+resistance+artery+function+using+pressure+myography.">Assessing murine resistance artery function using pressure myography.</a> J Vis Exp. (76), (2013).</li></ol>

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?...

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><tbody><tr><td>&lt;strong&gt;Chemical Name&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetylcholine</td><td>Sigma Aldrich</td><td>A6625</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>223506</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G5767</td><td></td></tr><tr><td>Endothelin-1</td><td>Sigma Aldrich</td><td>E7764</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetra acetic acid (EGTA)</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylene diamine tetra acetic acid (EDTA)</td><td>Sigma Aldrich</td><td>E9884</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3784</td><td></td></tr><tr><td>N&lt;sub&gt;&lt;em&gt;w&lt;/em&gt;&lt;/sub&gt;-nito-L-arginine methyl ester hydrochloride</td><td>Sigma Aldrich</td><td>N5751</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>Potassium chloride (KCL)</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Papaverine</td><td>Sigma Aldrich</td><td>P3510</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt; )</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH2Po4)</td><td>Sigma Aldrich</td><td>S9638</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Finescience tools</td><td>15000-08</td><td></td></tr><tr><td>Inverted microscope</td><td>Zeiss Achromat</td><td></td><td></td></tr><tr><td>Laboratory tubing</td><td>Euro-Pharm</td><td>250100306F999</td><td></td></tr><tr><td>Needle/pippette puller</td><td>David kopf instruments</td><td>720</td><td></td></tr><tr><td>Ophthalmic monofilament nylon suture</td><td>Surgical specialties</td><td>A7756N</td><td></td></tr><tr><td>Scissors</td><td>Finescience tools</td><td>150000-08</td><td></td></tr><tr><td>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) ?...

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

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

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

方法论方法血管功能和形态的非侵入性评估

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

免疫与感染

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

微血管碎片血管化产热脂肪组织的三维培养

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

生物工程

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

生物学

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

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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 ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent 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 ...