Department of Biomedicine, Hematology Section, University of Bergen, Jonas Lies Vei 91, Bergen, Norway.
Am J Physiol Heart Circ Physiol. 2012 Jan;302(2):H391-401. doi: 10.1152/ajpheart.00842.2011. Epub 2011 Nov 18.
There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.
目前缺乏非侵入性定量检测小型实验动物淋巴管功能的方法，而这是研究淋巴管系统病理生理学的必要条件。我们提出了一种新的方法，可以定量检测小鼠和大鼠的淋巴流量，该方法基于光学监测近红外荧光标记白蛋白的储库清除，并随后计算清除率常数（k）。BSA 与 Alexa680 NHS 酯缀合，在富含蛋白质的溶液中保持稳定，没有游离染料解离。为了评估淋巴流量，在皮内注射 0.5 或 1 μl Alexa680-白蛋白后，每隔 30 或 60 分钟对小鼠或大鼠进行成像，持续 3 至 6 小时。在测量过程中，小鼠保持清醒，而大鼠在整个实验过程中处于麻醉状态。k 是一个参数，定义为与淋巴流量等效，从所得对数线性洗脱曲线的斜率计算得出，对照 C57BL/6 和 C3H 小鼠的 k 值分别为-0.40 ± 0.03 和-0.30 ± 0.02%/min。在小鼠中局部给予血管收缩剂内皮素-1 会导致 k 值显著降低，而在大鼠中过度水合会增加 k 值，这反映了毛细血管滤过和淋巴流量之间的耦合。此外，Chy 型淋巴水肿小鼠（其皮肤淋巴管缺失）的 k 值为野生型的 50%。我们得出结论，通过光学成像检测亚微升数量的 Alexa680-白蛋白的储库清除，可以确定淋巴流量及其速率常数 k。该方法为评估小鼠和大鼠的淋巴管功能提供了一种微创、可重复且简单的替代方法。
