Vascular permeability of skeletal muscle microvessels in rat arterial ligation model: in vivo analysis using two-photon laser scanning microscopy

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 mu m(3) x 10(6)), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 +/- 0.40 mu m(3) x 10(6)) and 7 (2.5 +/- 0.8 mu m(3) x 10(6)) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 +/- 0.06 mu m) and 7 (0.21 +/- 0.08 mu m) days versus control (0.11 +/- 0.03 mu m, both P < 0.05). Capillary endothelial cell swelling was temporally and quanti-tatively 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.