激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具
Summary
胰岛微血管血管舒缩调节胰岛血液分布, 维持胰岛β细胞的生理功能。本协议描述了使用激光多普勒显示器测定胰岛微血管血管舒缩在体内的功能状态, 并评估胰岛微循环对胰腺相关疾病的贡献。
Abstract
微血管血管舒缩作为微循环的功能性状态, 对于氧气和养分的输送以及二氧化碳和废弃物的去除具有重要意义。微血管血管舒缩的损伤可能是微循环相关疾病发展的关键步骤。此外, 高血管化胰岛适应内分泌功能。在这方面, 似乎可以推断胰岛微血管血管舒缩的功能状态可能影响胰岛功能。胰岛微血管血管舒缩功能状态的病理变化分析可能是确定胰岛微循环对相关疾病如糖尿病的贡献的可行策略,胰腺炎,等。因此, 本协议描述使用激光多普勒血流监测仪确定胰岛微血管血管舒缩的功能状态, 建立参数 (包括平均血流灌注、振幅、频率和相对胰岛微血管血管舒缩的速度, 用于评价微循环功能状态。在链脲佐菌素诱导的糖尿病小鼠模型中, 我们观察到胰岛微血管血管舒缩功能状态受损。总之, 这种评估胰岛微血管血管舒缩在体内的方法可能揭示与胰岛疾病有关的机制。
Introduction
微血管血管舒缩作为微循环功能状态的一个参数, 负责氧气、营养素和荷尔蒙的传递和交换, 对去除代谢产物, 如二氧化碳和细胞浪费至关重要。1. 微血管血管舒缩还调节血液流量分布和组织灌注, 从而影响局部微循环血压和炎症反应, 可诱发多种疾病的水肿。因此, 微血管血管舒缩对维持器官的生理功能非常重要2, 3, 4, 组织和组件单元格.微血管血管舒缩的损害可能是发展微循环相关疾病的关键步骤之一5。
激光多普勒初步发展为观察和定量在微循环研究领域的6。该技术与其他技术方法 (例如,激光散斑7, 经皮氧,等) 一起被认为是评价微循环血流的黄金标准。根据多普勒移位原理, 用激光多普勒装置测定局部微循环 (即,毛细血管, 动脉, 静脉,等) 的血流灌注的基本原理。当光粒子遇到微血管中的运动血细胞时, 受激发射光的波长和频率会发生变化, 或者保持不变。因此, 在微循环中, 血细胞的数量和速度是与多普勒转移光的大小和频率分布有关的关键因素, 而微血管血流方向则无关紧要。使用不同的方法, 各种组织已被用于微循环研究, 包括 mesenteries 和背皮脂室的小鼠, 大鼠, 仓鼠, 甚至人类8。然而, 在本协议中, 我们重点研究胰岛微血管血管舒缩的功能状态, 用激光多普勒和自制的评价参数系统进行评价。
胰岛微循环主要由胰岛微血管构成, 表现出鲜明的特征。胰岛毛细血管网络显示五倍以上的密度比毛细管网络的外分泌对应9。胰岛内皮细胞提供输入葡萄糖和传播胰岛素的导管, 将氧气传递给胰岛β细胞代谢活性细胞。此外, 新的证据也表明, 胰岛微血管不仅涉及调节胰岛素基因表达和β细胞生存, 而且还影响β细胞功能;促进β细胞增殖;并产生一些血管活性、血管生成物质和生长因子10。因此, 在这方面, 我们推断胰岛微血管血管舒缩的功能状态可能影响胰岛β细胞功能, 并参与疾病的发病机制, 如急性/慢性胰腺炎, 糖尿病等与胰腺有关的疾病。
分析胰岛微血管血管舒缩功能状态的病理变化可能是确定胰岛微循环对上述疾病贡献的可行策略。详细的分步过程描述的方法, 以确定胰岛微血管血管舒缩在体内提供这里。然后, 将在代表性结果中显示典型的度量值。最后, 在讨论中突出显示方法的优点和限制, 以及进一步的应用程序。
Protocol
所有动物实验都是按照所有相关的指导方针、法规和监管机构执行的。目前正在示范的这项议定书是在北京协和医学院 (PUMC) 的微循环动物伦理委员会 (IMAEC) 的指导和批准下进行的。 1. 动物 实验开始前, 在 5%-小时的光暗循环下, 保持每笼三 BALB 小鼠, 控制温度 (24 1 摄氏度) 和湿度 (55 @ 12)。允许老鼠免费获得正常的食物和水。 将小鼠随机分为非糖尿病对照组和糖…
Representative Results
在图 1A中显示了配备半导体激光二极管的微血管血管舒缩测量激光多普勒装置的照片。用户界面软件显示在图 1B中。采用上述方法, 对非糖尿病对照和糖尿病小鼠胰岛微血管血管舒缩的血流动力学参数进行检测。许多技术, 包括激光多普勒血流仪, 反射和散射光, 红外光谱和成像技术, 已被用来研究微血管血管舒缩, 因为它是首次?…
Discussion
在涉及微血管功能障碍的病例中 (例如,糖尿病、急性胰腺炎、周围微血管疾病、等), 一些疾病导致血流减少。除了血液流动的变化, 还有重要的指标, 如微血管血管舒缩, 反映微循环的功能状态。具体指标, 微血管血管舒缩, 一般被定义为微血管的音调在微血管床上的振荡。在本协议中, 微血管血流灌注监测系统允许对微血管血管舒缩的功能状态进行直接的可视化和定量分析。我们的微?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了北京协和医学院青年基金和中央大学基础研究基金的资助 (3332015200 号赠款)。
Materials
| MoorVMS-LDF2 | Moor Instruments | GI80 | PeriFlux 5000 (Perimed Inc.) can be used as an alternative apparatus to harvest data |
| MoorVMS-PC Software | Moor Instruments | GI80-1 | Software of MoorVMS-LDF2 |
| Calibration stand | Moor Instruments | GI-cal | Calibration tool |
| Calibration base | Moor Instruments | GI-cal | Calibration tool |
| Calibration flux standard | Moor Instruments | GI-cal | Calibration tool |
| One Touch UltraEasy glucometer | Johnson and Johnson | #1955685 | Confirm hyperglycemia |
| One Touch UltraEasy strips | Johnson and Johnson | #1297006 | Confirm hyperglycemia |
| Streptozotocin | Sigma-Aldrich | S0130 | Dissolve in sodium citrate buffer (pH 4.3) |
| Pentobarbital sodium | Sigma-Aldrich | P3761 | Working concentration 3 % |
| Ethanol | Sinopharm Inc. | 200121 | Working concentration 75 % |
| Sucrose | Amresco | 335 | Working concentration 10 % |
| Medical gauze | China Health Materials Co. | S-7112 | Surgical |
| Blunt-nose forceps | Shang Hai Surgical Instruments Inc. | N-551 | Surgical |
| Surgical tapes | 3M Company | 3664CU | Surgical |
| Gauze sponge | Fu Kang Sen Medical Device CO. | BB5447 | Surgical |
| Scalpel | Yu Lin Surgical Instruments Inc. | 175C | Surgical |
| Skin scissor | Carent | 255-17 | Surgical |
| Suture | Ning Bo Surgical Instruments Inc. | 3325-77 | Surgical |
| Syringe and 25-G needle | MISAWA Inc. | 3731-2011 | Scale: 1 ml |
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Citazione di questo articolo
Liu, M., Zhang, X., Li, B., Wang, B., Wu, Q., Shang, F., Li, A., Li, H., Xiu, R. Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo. J. Vis. Exp. (133), e56028, doi:10.3791/56028 (2018).