Shimotsu Rie, Hotta Kzuki, Ikegami Ryo, Asamura Tomoyo, Tabuchi Ayaka, Masamoto Kazuto, Yagishita Kazuyoshi, Poole David C, Kano Yutaka
Department of Engineering Science, University of Electro-Communications, Chofu, Japan.
Department of Physical Therapy, Niigata University of Health and Welfare, Niigata, Japan.
Am J Physiol Regul Integr Comp Physiol. 2021 Jun 1;320(6):R972-R983. doi: 10.1152/ajpregu.00135.2020. Epub 2021 May 5.
Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 μm × 10), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 μm × 10) and 7 (2.5 ± 0.8 μm × 10) days was increased (both < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 μm) and 7 (0.21 ± 0.08 μm) days versus control (0.11 ± 0.03 μm, both < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.
下肢外周动脉疾病（PAD）由于动脉闭塞而导致氧气供应受损。缺血性骨骼肌伴有毛细血管结构变形。因此，我们使用新型显微镜技术，检验了内皮细胞肿胀在时间和数量上与微血管通透性增强相对应的假说。通过结扎雄性Wistar大鼠的髂动脉（AL）制造后肢缺血模型。在结扎后3天和7天，通过静脉注射异硫氰酸罗丹明B葡聚糖荧光染料，利用双光子激光扫描显微镜（TPLSM）对胫前（TA）肌微循环进行成像，并对染料外渗进行定量，以评估微血管通透性。通过透射电子显微镜（TEM）分析TA肌微血管内皮超微结构。与对照组（0.40±0.15μm×10）相比，使用TPLSM测量，在3天（3.0±0.40μm×10）和7天（2.5±0.8μm×10）时，荧光染料的体积测定间质渗漏增加（均P<0.05）。与对照组（0.11±0.03μm）相比，在3天（0.21±0.06μm）和7天（0.21±0.08μm）时，毛细血管壁厚度也增加（均P<0.05）。在PAD的AL模型中，毛细血管内皮细胞肿胀在时间和数量上与血管通透性升高相关，但这些变化发生时，血管内皮生长因子（VEGF）及其受体（VEGFR2，在AL术后7天降低）或基质金属蛋白酶的蛋白水平并未升高。内皮细胞肿胀与血管通透性增加的时间一致性支持存在一个共同的上游介质。TPLSM与TEM相结合，提供了一种灵敏且空间分辨的技术，用于评估针对外周动脉功能受损的有害后遗症的治疗对策的作用机制和疗效。
