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Background After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. Methods To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. Results Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-β1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. Conclusion Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

Introduction BALB/c mice and C57/BL6 mice have different abilities to recover from ischemia. C57/BL6 mice display increased vessel collateralization and vascular endothelial growth factor expression with a consequent rapid recovery from ischemia compared with BALB/c mice. Mesenchymal stem cells (MSCs) are one of the main cell types that contribute to the recovery from ischemia because, among their biological activities, they produce several proangiogenic paracrine factors and differentiate into endothelial cells. The objective of this study was to evaluate whether the MSCs of these two mouse strains have different inductive capacities for recovering ischemic limbs. Methods MSCs from these two strains were obtained from the bone marrow, purified and characterized before being used for in vivo experiments. Limb ischemia was surgically induced in BALB/c mice, and MSCs were injected on the fifth day. The evolution of limb necrosis was evaluated over the subsequent month. Muscle strength was assessed on the 30th day after the injection, and then the animals were sacrificed to determine the muscle mass and perform histological analyses to detect cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. Results The MSCs from both strains promoted high level of angiogenesis similarly, resulting in good recovery from ischemia. However, BALB/c MSCs promoted more muscle regeneration (57%) than C57/BL6 MSCs (44%), which was reflected in the increased muscle strength (0.79 N versus 0.45 N). Conclusion The different genetic background of MSCs from BALB/c mice and C57/BL6 mice was not a relevant factor in promoting angiogenesis of limb ischemia, because both cells showed a similar angiogenic activity. These cells also showed a potential myogenic effect, but the stronger effect promoted by BALB/c MSCs indicates that the different genetic background of MSCs was more relevant in myogenesis than angiogesis.