In vivo Ca2+ dynamics during cooling after eccentric contractions in rat skeletal muscle.

The effect of cooling on in vivo intracellular calcium ion concentration [Ca2+]i after eccentric contractions (ECs) remains to be determined. We tested the hypothesis that cryotherapy following ECs promotes an increased [Ca2+]i and induces greater muscle damage in two muscles with substantial IIb and IIx fiber populations. The thin spinotrapezius (SPINO) muscles of Wistar rats were used for in vivo [Ca2+]i imaging, and tibialis anterior (TA) muscles provided greater fidelity and repeatability of contractile function measurements. SPINO [Ca2+]i was estimated using fura 2-AM and the magnitude, location, and temporal profile of [Ca2+]i determined as the temperature near the muscle surface post-ECs was decreased from 30°C (control) to 20°C or 10°C. Subsequently, in the TA, the effect of post-ECs cooling to 10°C on muscle contractile performance was determined at 1 and 2 days after ECs. TA muscle samples were examined by hematoxylin and eosin staining to assess damage. In SPINO, reducing the muscle temperature from 30°C to 10°C post-ECs resulted in a 3.7-fold increase in the spread of high [Ca2+]i sites generated by ECs (P < 0.05). These high [Ca2+]i sites demonstrated partial reversibility when rewarmed to 30°C. Dantrolene, a ryanodine receptor Ca2+ release inhibitor, reduced the presence of high [Ca2+] sites at 10°C. In the TA, cooling exacerbated ECs-induced muscle strength deficits via enhanced muscle fiber damage (P < 0.05). By demonstrating that cooling post-ECs potentiates [Ca2+]i derangements, this in vivo approach supports a putative mechanistic basis for how postexercise cryotherapy might augment muscle fiber damage and decrease subsequent exercise performance.

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 µm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 µm3 × 106) and 7 (2.5 ± 0.8 µm3 × 106) 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) days 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.