Abstract
BackgroundAfter 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.MethodsTo 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.ResultsMuscle 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 < 0.01) on day 30. uP-mGM electroporation also increased 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.ConclusionCollectively, 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.
Highlights
Muscle injuries often require medical care and are difficult to treat, resulting in absences from and disabilities at work and causing a significant public health burden [1]
The expression of Gm-csf and its receptors was analyzed by RT-qPCR
When tibialis anterior (TA) muscles were subjected to both injury and uP vector expressing murine GM-CSF alone (GM)-CSF (uP-mGM) electroporation (LM+GM), the Gmcsf expression profile was similar to that of the GM group, but it lasted longer
Summary
Muscle injuries often require medical care and are difficult to treat, resulting in absences from and disabilities at work and causing a significant public health burden [1]. The healing process in the injured skeletal muscle consists of overlapping phases of degeneration, inflammation, regeneration, and fibrosis [2, 6]. Efficient regeneration of the injured muscle is thought to compete with fibrotic healing, and excessive fibrosis is thought to impede regeneration. This balance depends mainly on the cells and factors that are present at the degeneration and inflammation stages of healing. After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. 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
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