Department of Rehabilitation Sciences, Graduate School of Medical Sciences, Kitasato University, Sagamihara, Japan.
Department of Rehabilitation, Kitasato University School of Allied Health Sciences, Sagamihara, Japan.
J Appl Physiol (1985). 2024 Oct 1;137(4):963-974. doi: 10.1152/japplphysiol.00222.2024. Epub 2024 Aug 15.
This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in the skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats ( = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in tibialis anterior (TA) of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm × min × 10; endothelial thickness, 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter, 6.3 ± 0.80 vs. 13.6 ± 1.7/100 µm; cleft width, 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA, respectively, all < 0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared with the CON group ( < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles. Microvascular permeability in diabetic muscle was investigated using our original two-photon scanning laser microscopy method. Compared with controls, the leakage volume was increased in diabetic muscle, which was atrophic with smaller capillary diameter, endothelial cell thickening, and the appearance of more endothelial intercellular gaps or clefts, and large vesicles. Hyperpermeability was closely related to ultrafine structural changes of the capillary endothelial cell junctions.
本研究通过双光子激光扫描显微镜（TPLSM）和透射电子显微镜（TEM）观察糖尿病（DIA）大鼠骨骼肌毛细血管的微血管通透性和超微结构。采用活体成像技术，经静脉注射荧光染料后，评估雄性 Wistar 大鼠（CON 和 DIA[链脲佐菌素]，每组 20 只，10-14 周）比目鱼肌的微血管通透性。通过定量荧光染料渗漏来确定微血管通透性。通过 TEM 对离体骨骼肌进行超微结构成像，计算毛细血管内皮细胞之间的细胞间隙大小和数量以及细胞内囊泡。与对照组相比，糖尿病大鼠比目鱼肌的间质荧光染料渗漏量、内皮细胞厚度和每毛细血管周长的细胞间隙数显著增加，细胞间隙宽度显著变窄（间质荧光染料渗漏量，2.88±1.40 比 10.95±1.41 µm×min×10；内皮细胞厚度，0.28±0.02 比 0.45±0.03 µm；每毛细血管周长的细胞间隙数，6.3±0.80 比 13.6±1.7/100 µm；细胞间隙宽度，11.92±0.95 比 8.40±1.03 nm，CON 比 DIA，均<0.05）。与对照组相比，糖尿病组血管内皮细胞内囊泡的大小显示大囊泡的比例增加（<0.05）。糖尿病导致骨骼肌微血管的微血管通透性增加，部分原因是内皮细胞间隙的密度增加和变窄，以及更大的细胞内囊泡。我们采用原创的双光子扫描激光显微镜方法研究了糖尿病肌肉的微血管通透性。与对照组相比，糖尿病肌肉的渗漏量增加，其特征为毛细血管直径较小、内皮细胞增厚、出现更多内皮细胞间隙或裂孔以及大囊泡。通透性增加与毛细血管内皮细胞连接的超微结构变化密切相关。
