Regeneration Of Injured Muscle Research Articles

M2 Macrophage-derived exosomal miR-501 contributes to pubococcygeal muscle regeneration

Pubococcygeal muscle injury can lead to stress urinary incontinence (SUI). M2 macrophages play a crucial role in myoblast differentiation during injured muscle regeneration. However, the underlying mechanism remains unclear. Recently, exosomes have attracted increasing attention due to their mediation of cell-to-cell communication. In this study, we found that M2 macrophages extensively infiltrated the pubococcygeal muscle on day 5 after injury (VD5) in vivo. Then, C2C12 myoblasts were treated with M2 macrophage-derived exosomes (M2-EXO) and the results revealed that these exosomes could promote myotube formation. MiR-501 was identified as one of the abundant microRNAs (miRNAs) selectively loaded in M2-EXO, and subsequently confirmed to promote C2C12 myoblast differentiation by targeting YY1. Moreover, in vivo experiments showed that M2-EXO improves the inflammatory cell infiltration and have a therapeutic effect on damaged pubococcygeal muscle in SUI models. Collectively, our present results provide new insights into the promyogenic mechanism of M2 macrophages and prove that M2 macrophage exosomal miR-501 may represent a potential therapeutic to promote recovery from diseases caused by muscle injury, including SUI.

The importance of pseudocapsule preservation during hysteroscopic myomectomy

Minimally invasive techniques for myomectomy are based on the rationale of preserving the myometrial integrity, in order to spare muscular and fibro-neurovascular myometrial fibers and ensure complete and bloodless myoma removal. Post-operative myometrial vascularization is crucial in injured muscle regeneration. The post-surgical myometrial healing is needful for uterine reproductive function. Neurotransmitters and neurofibers were analyzed in the myoma pseudocapsule surrounding fibroid. They activate signaling molecule synthesis and release which, in turn, promote cell activation and induce muscle regeneration and growth. Pseudocapsule damage during myomectomy may lead to a reduction of neuropeptides and neurofibers at the hysterotomic site, to a poor physiological myometrial healing, with more fibrosis due to hypoxia, ischemia and necrosis.These pathophysiological events cause deficit in myometrial neurotransmission, muscular impulse and contractility, with ultimately impaired uterine muscle function during pregnancy, labor and delivery. Hence, during myomectomy, all manipulations should be performed as precisely and bloodlessly as possible, avoiding extensive, high wattage diathermocoagulation or excessive tissue manipulation or muscular trauma. Any iatrogenic pseudocapsule damage may alter neurotransmitter function during successive myometrial healing, impacting negatively on uterine repair and on eventual pregnancies. Hence the reasoned myomectomy on a biological basis, the “intracapsular myomectomy”, satisfied these surgical and physiological requirements. It was described precisely and firstly by the hysteroscopy, with the image magnification of the preservation of the myoma pseudocapsule. The “intracapsular hysteroscopic myomectomy” demonstrated the safe and effective removal of submucous myomas with intramural development. It allowed to completely remove the myoma in one or two surgical steps, saving the pseudocapsule and the surrounding healthy myometrium. The respect of the myometrium and the reduced thermal injury, a part the excellent outcomes in terms of surgical complications prevention, post-surgical fibrosis and intrauterine synechiae reduction, highlighted the physiological development of a successive pregnancy, without any myometrial complications during pregnancy, labor and delivery.

Biotoxins in muscle regeneration research.

Skeletal muscles are characterized by their unique regenerative capacity following injury due to the presence of muscle precursor cells, satellite cells. This characteristic allows researchers to study muscle regeneration using experimental injury models. These injury models should be stable and reproducible. Variety of injury models have been used, among which the intramuscular injection of myotoxic biotoxins is considered the most common and widespread method in muscle regeneration research. By using isolated biotoxins, researchers could induce acute muscle damage and regeneration in a controlled and reproducible manner. Therefore, it is considered an easy method for inducing muscle injury in order to understand the different mechanisms involved in muscle injuries and tissue response following injury. However, different toxins and venoms have different compositions and subsequently the possible effects of these toxins on skeletal muscle vary according to their composition. Moreover, regeneration of injured muscle by venoms and toxins varies according to the target of toxin or venom. Therefore, it is essential for researcher to be aware of the mechanism and possible target of toxin-induced injury. The current paper provides an overview of the biotoxins used in skeletal muscle research.

Platelet-Rich Plasma Releasate Promotes Regeneration and Decreases Inflammation and Apoptosis of Injured Skeletal Muscle

Background: Platelet-rich plasma (PRP) contains various cytokines and growth factors that may be beneficial to the healing process of injured muscle. Based on the authors’ previous study, PRP releasate can promote proliferation and migration of skeletal muscle cells in vitro, so animal studies are performed to support the use of PRP to treat muscle injury in vivo. Purpose: To investigate the effect of PRP releasate on regeneration of injured muscle, as well as its effect on inflammatory reaction and cell apoptosis, in the early stages of the muscle-healing process. Study Design: Controlled laboratory study. Methods: The gastrocnemius muscles of Sprague-Dawley rats were injured by partial transverse incision and then treated with PRP releasate. Hematoxylin and eosin stain was used to evaluate the healing process of injured muscle at 2, 5, and 10 days after injury. TUNEL assay was used to evaluate the cell apoptosis of injured muscle after PRP releasate treatment. Immunohistochemistry was used to stain the CD68-positive cells during the healing process. Muscle contractile properties, including fast-twitch and tetanic strength, were evaluated by electric stimulation. Results: The results revealed that PRP releasate treatment could enhance the muscle-healing process and decrease CD68-positive cells and apoptotic cells. Furthermore, the tetanic strength was significantly higher in injured muscle treated with PRP releasate. Conclusion: In conclusion, PRP releasate could enhance the healing process of injured muscle and decrease inflammatory cell infiltration as well as cell apoptosis. Clinical Relevance: PRP promotes skeletal muscle healing in association with decreasing inflammation and apoptosis of injured skeletal muscle. These findings provide in vivo evidence to support the use of PRP to treat muscle injury.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

Physical activity and alpha-lipoic acid modulate inflammatory response through changes in thiol redox status

α-Lipoic acid (αLA), as an inductor of hydrogen peroxide (H2O2) and nitrogen oxide (NO) generation and modulator of thiol redox status, plays an important role in cell signalling pathways. The study was designed to observe the effect of αLA on inflammatory response through changes in H2O2 and NO levels as well as thiol redox status. Sixteen physically active males were randomly assigned to one of two groups: placebo or αLA (1,200 mg d(-1) for 10 days prior to exercise). The exercise trial involved a 90-min run at 65% VO2max (0% gradient) followed by 15-min eccentric phase at 65% VO2max (-10% gradient). Blood samples were collected before the exercise trial and then again 20 min, 24, and 48 h after. αLA significantly elevated H2O2 but reduced NO generation before or after exercise. Thiol redox status (GSHtotal-2GSSG/GSSG) increased by >50% after αLA and exercise (ANOVA, P < 0.05) and correlated with changes in cytokines interleukin-6 (IL-6) (r = -0.478, P < 0.05) and IL-10 (r = -0.455, P < 0.05). This was caused by strong effect of αLA on GSSG concentration. αLA elevated IL-6 and IL-10 levels at 20 min after exercise and decreased in interleukin-1β and tumor necrosis factor α before and after exercise. This enhanced the regeneration of injured muscles. Creatine kinase activity tended to lower values after αLA intake. The study suggests that the combination of intense exercise with α-lipoic acid intake might be useful to improve the skeletal muscle regeneration through changes in inflammatory response which are associated with H2O2 and NO generation as well as thiol redox status.

Role of Integrin-β3 Protein in Macrophage Polarization and Regeneration of Injured Muscle

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-β3 was initially expressed, mainly in macrophages. In integrin-β3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-β3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-β1 and increased TGF-β1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-β3 produced more TGF-β1. Furthermore, transplantation of bone marrow cells from integrin-β3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-β3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-β1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-β3 function could stimulate muscle regeneration.

Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.

Administration of granulocyte colony-stimulating factor facilitates the regenerative process of injured mice skeletal muscle via the activation of Akt/GSK3αβ signals

Effects of administration of granulocyte colony-stimulating factor (G-CSF) on the regeneration of injured mammalian skeletal muscles were studied in male C57BL/6J mice. Muscle injury was induced by injection of cardiotoxin (CTX) into tibialis anterior muscles bilaterally. G-CSF was administrated for 8 consecutive days from 3 days before and 5 days after the injection. Significant decreases of wet weight and protein content were noted in the necrotic muscle with CTX injection. A large number of the regenerating fibers having central nucleus were observed 7 days after the injection. The regeneration of injured muscle was further facilitated by the G-CSF treatment. Population of Pax7-positive nuclei was increased by the G-CSF treatment at day 7. Phospho-Akt and phospho-glycogen synthase kinase 3alphabeta (GSK3alphabeta) signals were also activated by G-CSF-administrated group during the regenerative process. It was suggested that G-CSF treatment may facilitate the regeneration of injured skeletal muscles via the activation of Akt/GSK3alphabeta signals.

Inhibited skeletal muscle healing in cyclooxygenase-2 gene-deficient mice: the role of PGE2and PGF2α

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat skeletal muscle injury. However, studies have shown that NSAIDs may be detrimental to the healing process. Mediated by prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)), the cycloxygenase-2 (COX-2) pathway plays an important role in muscle healing. We hypothesize that the COX-2 pathway is important for the fusion of muscle cells and the regeneration of injured muscle. For the in vitro experiments, we isolated myogenic precursor cells from wild-type (Wt) and COX-2 gene-deficient (COX-2(-/-)) mice and examined the effect of PGE(2) and PGF(2alpha) on cell fusion. For the in vivo experiments, we created laceration injury on the tibialis anterior (TA) muscles of Wt and COX-2(-/-) mice. Five and 14 days after injury, we examined the TA muscles histologically and functionally. We found that the secondary fusion between nascent myotubes and myogenic precursor cells isolated from COX-2(-/-) mice was severely compromised compared with that of Wt controls but was restored by the addition of PGF(2alpha) or, to a lesser extent, PGE(2) to the culture. Histological and functional assessments of the TA muscles in COX-2(-/-) mice revealed decreased regeneration relative to that observed in the Wt mice. The findings reported here demonstrate that the COX-2 pathway plays an important role in muscle healing and that prostaglandins are key mediators of the COX-2 pathway. It suggests that the decision to use NSAIDs to treat muscle injuries warrants critical evaluation because NSAIDs might impair muscle healing by inhibiting the fusion of myogenic precursor cells.

A Gene That Directs the Regeneration of Injured Muscle from Adult Stem Cells

A Gene That Directs the Regeneration of Injured Muscle from Adult Stem Cells

Effect of pulsed laser radiation on regeneration of injured muscles with different regeneration capacities and the state of the thymus.

Effect of pulsed laser radiation on regeneration of injured muscles with different regeneration capacities and the state of the thymus.

Muscle strain injuries.

Muscle injuries--lacerations, contusions or strains--are by far the most common injuries in sports. After first aid following the RICE principle (Rest, Ice, Compression and Elevation), therapy must be tailored according to the severity of the injury and based on the knowledge gained from experimental studies on regeneration of injured muscle. Most muscle injuries can be treated conservatively with excellent recovery, but complete ruptures with complete loss of function should be managed surgically. Immediately after the injury, a short period of immobilization is needed to accelerate formation of the scar between the stumps of the ruptured myofibers, to which the stumps adhere. The optimal length of immobilization depends on the grade of the injury, and should not be longer than needed for the scar to bear the pulling forces without rerupture. Early mobilization is required to invigorate adhesion, orientation of the regenerating muscle fibers, revascularization and resorption of the connective tissue scar. Another important aim of early mobilization, especially in clinical sports medicine, is to minimize inactivity-induced atrophy as well as loss of strength and extensibility, which are rapidly appearing adverse sequelae of prolonged immobilization.

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I producing human T cells.

A thymus-derived myoid precursor cell line (ST1), which differentiates to myoid cells in the growth arrest condition, was established by the cocultivation of F344 rat thymic cells with human T-lymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. No integration of HTLV-I was detected in ST1 cells by Southern blot hybridization. In a differentiation culture condition such as confluent culture or serum starvation, ST1 cells began to fuse, creating multinuclear giant cells, with the induced expression of MyoD1 and various muscle-specific antigens, including alpha-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin, and acetylcholine receptor. Ultrastructural investigation revealed that differentiated ST1B cells created aggregates of thick and thin filaments with Z-band-like composition, then formed sarcomeric structures and tubular honeycomb arrays. Finally, these cells spontaneously contracted with a frequency of 0.5-2.0 Hz and synchronized with adjoining cells. Transplantation of ST1B cells into nude mice produced a small tumor nodule, showing clear differentiation to skeletal muscle cells. ST1B cells did not indicate any colony-forming activities in soft agar, demonstrating that ST1B cells retain some of the physiologically normal phenotypes. This rare cell line is promising for use in various physiological and pathological investigations including functional research of thymic myoid cells and the pathological role in autoimmune diseases, as well as animal model experiments of cell therapy related to muscular degenerative disorders or regeneration of injured muscles.

The role of leukemia inhibitory factor in skeletal muscle regeneration.

Although a number of cytokines have been implicated in tissue regeneration, it is unknown which ones actually function in vivo. Here, we use mice with a targeted mutation in the leukemia inhibitory factor (LIF) gene to examine the role of LIF in muscle regeneration. Using a muscle crush model, we show that muscle regeneration in LIF knockout mice is significantly, reduced compared to control littermates. Further, targeted infusion of LIF in both normal and LIF knockout animals stimulated muscle regeneration, but the stimulation observed was much greater in the mutant animals than in controls. In contrast, interleukin-6 and transforming growth factor-alpha, which also stimulate myoblast proliferation in vitro, had no effect on regeneration. These findings demonstrate directly that LIF is involved in regeneration of injured muscle and points to the use of LIF as a therapeutic agent in the treatment of neuromuscular disease.

Localization of myogenin, c-fos, c-jun, and muscle-specific gene mRNAs in regenerating rat skeletal muscle.

It has been suggested that myogenin is an important factor for the differentiation of myoblasts and that its function in myogenesis is regulated by proto-oncogenes in in vitro experiments. We have characterized the spatial and temporal expression patterns of myogenin, c-fos, c-jun, and muscle creatine kinase mRNAs during the skeletal muscle regeneration process using in situ hybridization histochemistry. Myogenin transcripts are first detected in the myonuclei/nuclei of satellite cells at 6 h after induction of regeneration. Myogenin mRNA is expressed in desmin-positive myoblasts, yet no muscle creatine kinase mRNA is detected in this cell type. Both the muscle creatine kinase and myogenin mRNAs are expressed in the newly formed myotubes, but not at earlier stages. Transcripts for c-fos and c-jun mRNAs are expressed first in the myonuclei/nuclei of satellite cells at 3 h post-trauma. c-jun mRNA is expressed in both myoblasts and myotubes, while c-fos mRNA was not detected in these cells. These results suggest that myogenin plays important role in the regeneration of injured muscle and that c-jun and c-fos may have different roles in this process.