Mizuno Risuke, Ono Nobuyuki, Ikomi Fumitaka, Ohhashi Toshio
Department of Physiology, Shinshu University School of Medicine, Matsumoto, Japan.
Lymphat Res Biol. 2005;3(3):127-36. doi: 10.1089/lrb.2005.3.127.
There is no reported study in which the lymphatic flow pathway in lymph nodes has been visualized and investigated in in vitro studies. The purpose of the present study is first to develop an isolated rat lymph node preparation circulated through the afferent and efferent lymph vessels in order to visualize lymphatic flow pathway within the node by using a video microscope system, and then to evaluate lymphatic flow dynamics by using fluorescence-labeled substances.
In the present study, rat iliac lymph node was isolated. The afferent and efferent lymph vessels of the lymph node were cannulated with glass micropipettes in an organ chamber, while the small-sized arteries and veins connected to the lymph node were secured with sutures. Krebs-bicarbonate solution with or without fluorescent probes [FITCdextran, mol. weight; 77,000 and/or fluoresbrite carboxylate-microspheres (FC-microspheres) mean diameter of 1 microm] was circulated through the lymph vessels of the node. The time-dependent lymphatic pathway of fluorescent probes was investigated with a video microscope system. FTIC-dextran first spread through the cortex and subsequently reached the medulla of the lymph node. A follicle-like structure became evident in the cortex. FITC-dextran appeared in the efferent lymph vessel at 109 +/- 21 sec after its circulation began, while FC-microspheres distributed in the lymph node did not emerge from the node within 20 min after their introduction.
In vitro preparations constructed in the present study will enable us to visualize the lymphatic flow pathway of fluorescent substances within the isolated iliac lymph nodes of rats in the absence of blood circulation.
在体外研究中，尚未有关于可视化和研究淋巴结中淋巴流动途径的报道。本研究的目的首先是开发一种通过输入淋巴管和输出淋巴管循环的离体大鼠淋巴结制剂，以便使用视频显微镜系统可视化淋巴结内的淋巴流动途径，然后使用荧光标记物质评估淋巴流动动力学。
在本研究中，分离出大鼠髂淋巴结。在器官腔室中用玻璃微量移液器插入淋巴结的输入淋巴管和输出淋巴管，同时用缝线固定与淋巴结相连的小动脉和小静脉。含有或不含荧光探针[FITC-葡聚糖，分子量；77,000和/或荧光羧酸盐微球（FC-微球），平均直径为1微米]的 Krebs-碳酸氢盐溶液通过淋巴结的淋巴管循环。用视频显微镜系统研究荧光探针的时间依赖性淋巴途径。FITC-葡聚糖首先扩散穿过皮质，随后到达淋巴结的髓质。皮质中出现了类似滤泡的结构。FITC-葡聚糖在其循环开始后109±21秒出现在输出淋巴管中，而引入淋巴结中的FC-微球在引入后20分钟内未从淋巴结中出现。
本研究构建的体外制剂将使我们能够在无血液循环的情况下可视化大鼠离体髂淋巴结内荧光物质的淋巴流动途径。
