Via modulation of endothelial integrity and vascular permeability in response to damage, skeletal muscle microvessels play a crucial permissive role in tissue leukocyte invasion. However, direct visual evidence of altered microvascular permeability of skeletal muscle has not been technically feasible, impairing mechanistic understanding of these responses. Two-photon laser scanning microscopy (TPLSM) allows three-dimensional in vivo imaging of skeletal muscle microcirculation. We hypothesized that the regulation of microvascular permeability in vivo is temporally related to acute inflammatory and regenerative processes following muscle injury. To test our hypothesis, tibialis anterior muscles of anesthetized male Wistar rats were subjected to eccentric contractions (ECCs) via electrical stimulation. The skeletal muscle microcirculation was imaged by an intravenously infused fluorescent dye (rhodamine B isothiocyanate-dextran) to assess microvascular permeability via TPLSM 1, 3, and 7 days after ECC. Immunohistochemistry on serial muscle sections was performed to determine the proportion of VEGF-A-positive muscle fibers in the damaged muscle. Compared with control rats, the volumetrically determined interstitial leakage of fluorescent dye (5.1 ± 1.4, 5.3 ± 1.2 vs. 0.51 ± 0.14 μm × 10; P < 0.05, days 1 and 3, respectively, vs. control) and percentage of VEGF-A-positive fibers in the damaged muscle (10 ± 0.4%, 22 ± 1.1% vs. 0%; days 1 and 3, respectively, vs. control) were significantly higher on days 1 and 3 after ECC. The interstitial leakage volume returned to control by day 7. These results suggest that microvascular hyperpermeability assessed by in vivo TPLSM imaging is associated with ECC-induced muscle damage and increased VEGF expression. NEW & NOTEWORTHY This investigation employed a novel in vivo imaging technique for skeletal muscle microcirculation using two-photon laser scanning microscopy that enabled microvascular permeability to be assessed by four-dimensional image analysis. By combining in vivo imaging and histological analysis, we found the temporal profile of microvascular hyperpermeability to be related to that of eccentric contraction-induced skeletal muscle injury and pronounced novel myocyte VEGF expression.
Recent studies have demonstrated the advantage of developing pressure-sensitive devices with light-emitting properties for direct visualization of pressure distribution, potential application to next generation touch panels and human-machine interfaces. To ensure that this technology is available to everyone, its production cost should be kept as low as possible. Here, simple device concepts, namely, pressure sensitive flexible hybrid electrodes and OLED architecture, are used to produce low-cost resistive or light-emitting pressure sensors. Additionally, integrating solution-processed self-assembled micro-structures into the flexible hybrid electrodes composed of an elastomer and conductive materials results in enhanced device performances either in terms of pressure or spatial distribution sensitivity. For instance, based on the pressure applied, the measured values for the resistances of pressure sensors range from a few MΩ down to 500 Ω. On the other hand, unlike their evaporated equivalents, the combination of solution-processed flexible electrodes with an inverted OLED architectures display bright green emission when a pressure over 200 kPa is applied. At a bias of 3 V, their luminance can be tuned by applying a higher pressure of 500 kPa. Consequently, features such as fingernails and fingertips can be clearly distinguished from one another in these long-lasting low-cost devices.
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 rats 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 μm3x106), using TPLSM the volumetrically-determined interstitial leakage of fluorescent dye measured at 3 (3.0±0.40 μm3x106) and 7 (2.5±0.8 μm3x106) days was increased (both P<0.05). Capillary wall thickness was also elevated at 3 (0.21±0.06 μm) and 7 (0.21±0.08 μm) versus control (0.11±0.03 μm, both p<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.
To investigate whether Type I diabetes (DIA) alters the temporal characteristics of muscle microvascular function and fiber damage incurred by eccentric contraction (ECC)‐induced muscle injury, male Wistar rats (n=53, 10–14 wks old) were randomly assigned to control (CON) or Streptozocin (45 mg/kg, i.p. DIA) groups. Four weeks later (blood glucose, 354±46 mg/dL), the tibialis anterior (TA) muscle of 1 leg was injured by 200 times ECC. Microvascular permeability was assessed by in vivo imaging using two‐photon laser scanning microscopy on 1, 3, 7 and 14 days post‐ECC. The TA microcirculation was visualized after intravenous infusion of fluorescent dye; extravasation of which was quantified to assess microvascular permeability. CON and ECC TA muscles were dissected and frozen after in vivo imaging, and serial cryosections were co‐stained with vascular endothelial growth factor A (VEGFA) antibody and hematoxylin‐eosin. Compared with CON rats, non‐ECC TA microvascular permeability was slightly but significantly enhanced in DIA with higher expression of VEGFA in myocytes. In CON rats, compared with non‐ECC TA, injured myocytes and microvascular hyper‐permeability were observed simultaneously on 1 and 3 days (injured myocyte 48, 73%, extravasation 262, 266 μm3×106xmin, 1, 3 days post‐ECC, respectively) and were repaired by 7 days after ECC (no injured myocytes, regenerated myocyte 29%, extravasation 101 μm3×106xmin). DIA TA exhibited delayed muscle injury onset and microvascular hyper‐permeability from 1 to between 3 and 7 days (injured 8, 81, 35%, extravasation 33, 183, 180 μm3×106xmin, 1, 3, 7 days post‐ECC, respectively). Regenerated myocyte was observed between 7 and 14 days post‐ECC (regenerated 47, 23%, 7, 14 days, respectively, extravasation 105 μm3×106xmin, 14 days post‐ECC). Regenerated cell size was smaller in DIA compared to CON (3214 vs. 1377 μm2, CON vs. DIA 14 days post‐ECC, P<0.01). Percentage of VEGFA‐positive myocytes to injured myocytes was increased in both CON (28%, 43%, 1 and 3 days post‐ECC, respectively) and DIA (44%, 43%, 65%, 1, 3, 7 days post‐ECC) rats with a temporal profile corresponding to that of increased dye extravasation. These results suggest that DIA retards the onset of microvascular hyper‐permeability after ECC thereby delaying skeletal muscle injury and recovery. VEGF may regulate microvascular permeability in both CON and DIA skeletal muscle and therefore play a commanding role in post‐ECC damage and regeneration.Support or Funding InformationThis work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP17K13185, 16700450, 18700526, and 23650403.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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