Skeletal Muscle Microvascular Permeability After Eccentric Contraction-Induced Muscle Injury

Abstract

Within injured skeletal muscle the capillary bed plays a crucial role in leukocyte invasion through modulations of the endothelial integrity, associated with increased permeability. However, direct observation of altered microvascular permeability and compromised capillary integrity has not been technically feasible. Two-photon laser scanning microscopy (TPLSM) allows three-dimensional in vivo imaging which, given the depth of penetration and high resolution of TPLSM, will facilitate measurement of microvascular leakage. PURPOSE: We hypothesized that the regulation of capillary permeability in vivo, as assessed by real-time TPLSM, is temporally related to acute inflammatory and regenerative processes following muscle injury. METHODS: Tibialis anterior muscles of anesthetized male Wistar rats (n=57) were subjected to eccentric contractions (ECC) via electrical stimulation. The skeletal muscle microcirculation was imaged by an intravenously infused fluorescent dye (rhodamine b isothiocyanate dextran, molecular weight 70,000 Daltons) to assess microvascular permeability via TPLSM 1, 3 and 7 days after ECC. Immunohistochemistry on serial muscle sections was performed to determine the percentage of VEGF-A positive muscle fibers in the damaged muscle. RESULTS: Compared with non-ECC control, the volumetrically-determined interstitial leakage of fluorescent dye had increased significantly on days 1 and 3 post-ECC (5.1±1.4, 5.3±1.2 vs. 0.51±0.14 μm3x106, P<0.05 respectively days 1 and 3 vs. control). However, by post-ECC day 7 interstitial leakage had returned to control values. Damaged muscle fibers were evident on days 1 and 3 (% damaged muscle fiber: 11.7±4.7, 48.4±12.4% vs. 0% P<0.05 respectively days 1 and 3 vs. control). Percentage of VEGF-A positive muscle fiber in damaged muscle fiber was significantly higher on days 1 and 3 compared to control (24.9±9.8, 39.3±16.7 vs. 0%, P<0.05 respectively days 1 and 3 vs. control). Regenerated skeletal muscle fibers were found only at 7 days post- ECC. CONCLUSION:In vivo TPLSM imaging represents a powerful investigative technique for skeletal muscle microcirculatory research. Microvascular hyperpermeability is associated with ECC-induced muscle damage and increased VEGF expression.