No abstract

Apart from its hematopoietic effect, erythropoietin (EPO) is known as pleiotropic cytokine with anti-inflammatory and antiapoptotic properties. Here, we evaluated for the first time the EPO-dependent regeneration capacity in an in vivo rat model of skeletal muscle trauma. A myoblast cell line was used to study the effect of EPO on serum deprivation-induced cell apoptosis in vitro. A crush injury was performed to the left soleus muscle in 80 rats treated with either EPO or saline. Muscle recovery was assessed by analysis of contraction capacities. Intravital microscopy, BrdU/laminin double immunohistochemistry and cleaved caspase-3 immunohistochemistry of muscle tissue on days 1, 7, 14, and 42 posttrauma served for assessment of local microcirculation, tissue integrity, and cell proliferation. Serum deprivation-induced myoblast apoptosis of 23.9 AE 1.5% was reduced by EPO to 17.2 AE 0.8%. Contraction force analysis in the EPO-treated animals revealed significantly improved muscle strength with 10-20% higher values of twitch and tetanic forces over the 42-day observation period. EPO-treated muscle tissue displayed improved functional capillary density as well as reduced leukocytic response and consecutively macromolecular leakage over day 14. Concomitantly, muscle histology showed significantly increased numbers of BrdU-positive satellite cells and interstitial cells as well as slightly lower counts of cleaved caspase-3-positive interstitial cells. EPO results in faster and better regeneration of skeletal muscle tissue after severe trauma and goes along with improved microcirculation. Thus, EPO, a compound established as clinically safe, may represent a promising therapeutic option to optimize the posttraumatic course of muscle tissue healing.

Insufficiency of skeletal muscle regeneration often impedes the healing process with functional deficiencies and scar formation. We tested the hematopoietic growth factor granulocyte-colony stimulating factor (G-CSF) with respect to its efficacy to improve functional muscle regeneration following skeletal muscle injury in Wistar rats. After crush injury to the left soleus muscle, animals received daily G-CSF (20 mug/kg ip) or vehicle solution (n = 30 per group each). Sham-operated animals without muscle injury served as controls (n = 15). After in vivo assessment of the fast-twitch and tetanic contraction capacity of the soleus muscles at days 4, 7, and 14 post-injury, sampling of muscle tissue served for analysis of satellite cell proliferation [bromodeoxyuridine (BrdU)/laminin and BrdU/desmin double immunohistochemistry] and cell apoptosis (transferase nick-end labeling analysis). Muscle strength analysis revealed recovery of contraction forces to 26 +/- 2, 35 +/- 3, and 53 +/- 3% (twitch force) and to 20 +/- 3, 24 +/- 2, and 37 +/- 2% (tetanic force) within the 14-day observation period in vehicle-treated animals. In contrast, G-CSF increased contractile forces with markedly higher values at day 7 (twitch force: 42 +/- 2%; tetanic force: 34 +/- 2%) and day 14 (twitch force: 62 +/- 3%; tetanic force: 43 +/- 3%). This enhancement of muscle function was preceded by a significant increase of satellite cell proliferation (BrdU-positive cells/mm(2): 27 +/- 6 vs. vehicle: 12 +/- 3) and a moderate decrease of cell apoptosis (transferase nick-end labeling-positive cells/mm(2): 11 +/- 2 vs. vehicle: 16 +/- 3) at day 4. In conclusion, G-CSF histologically promoted viability and proliferation of muscle cells and functionally enhanced recovery of muscle strength. Thus G-CSF might represent a therapeutic option to optimize the posttraumatic course of muscle tissue healing.

The goal of this study was to provide evidence that melatonin improves muscle healing following blunt skeletal muscle injury. For this purpose, we used 56 rats and induced an open muscle injury. After injury, all animals received either daily melatonin or vehicle solution intraperitoneally. Subsequent observations were performed at day 1, 4, 7, and 14 after injury. After assessment of fast twitch and tetanic muscle force, we analyzed leukocyte infiltration, satellite cell number, and cell apoptosis. We further quantified the expression of the melatonin receptor and the activation of extracellular-signal-regulated kinase (ERK). Chronic treatment with melatonin significantly increased the twitch and tetanic force of the injured muscle at day 4, 7, and 14. At day 1, melatonin significantly reduced the leukocyte infiltration and significantly increased the number of satellite cells when compared to the control group. Consistent with this observation, melatonin significantly reduced the number of apoptotic cells at day 4. Furthermore, phosphorylation of ERK reached maximal values in the melatonin group at day 1 after injury. Additionally, we detected the MT1a receptor in the injured muscle and showed a significant up-regulation of the MT1a mRNA in the melatonin group at day 4. These data support the hypothesis that melatonin supports muscle restoration after muscle injury, inhibits apoptosis via modulation of apoptosis-associated signaling pathways, increases the number of satellite cells, and reduces inflammation.

Insufficiency of skeletal muscle regeneration is often accompanied with functional deficiencies. The goal of our study was to assess the restoration of peripheral muscle upon injury of different severity. Blunt crush injury of the soleus muscle in rats was induced by a clamp and stepwise amplified in severity by rising the locking level of the clamp, resulting in three different groups (1Â lock; 2Â lock; 3Â lock; n ¼ 30 animals per group). After assessment of the fast twitch and tetanic contraction capacity at days 1, 4, 7, 14, and 42 postinjury sampling of muscle tissue served for analysis of cell proliferation, including satellite cells, apoptosis, and leukocyte infiltration. Contraction force analysis demonstrated significantly higher values of relative muscle strength in the 1Â lock group compared to the two other groups over 42 days. Calculation of the twitch-to-tetanic force ratio revealed significantly higher mean values at days 1, 7, and 14 in the animals of group 2Â lock and 3Â lock, indicating a transformation toward a fast-twitching muscular phenotype. Moreover, cell proliferation during the first 4 days was found dependent on the severity of muscle injury in that the higher the severity the higher the proliferation. At the same time, cell apoptosis was found increased, and at day 1 the local leukocyte infiltration was significantly higher in the 3Â lock compared to the 1Â lock group. These data indicate that severity of injury correlates with local repair responses, which, however, are not necessarily sufficient to fully restore muscle function. Keywords: proliferation; apoptosis; muscular biomechanics; bromodeoxyuridine; laminin Muscle injuries represent a challenging problem in current orthopedic surgery and traumatology. During the last 2 decades, soft tissue injury has increasingly been appreciated as a decisive factor for the patient's outcome and prognosis upon trauma.1 In accordance to the fact that skeletal muscle provides an important collateral source of blood to the cortical bone, 2 soft tissue damage significantly determines the process of bone healing and the guidance of fracture healing. 3,4 In addition, regain of function and duration of rehabilitation critically depends on the severity of soft tissue injury. Moderate muscle trauma after minor injuries frequently remains undiagnosed, but is responsible for chronic pain and restricted mobility. Extensive muscular lesions often cause livelong disability and marked reduction in quality of life. Functional limitations and incomplete rehabilitation present a further threat of disability as individuals attempt to interact with their social and physical environments. 5So far, no definitive treatment regimens after skeletal muscle injury exist. Novel experimental results only partially fulfill the scientist's expectations regarding muscle healing, and alternative approaches to enhance muscular regeneration might be necessary to develop convincing therapeutic strategies. 6 With this in mind, it is essential to better understand the pr...