Francis X. Pizza

Neutrophils contribute to muscle injury and impair its resolution after lengthening contractions in mice

We tested the hypotheses that: (1) neutrophil accumulation after contraction‐induced muscle injury is dependent on the β2 integrin CD18, (2) neutrophils contribute to muscle injury and oxidative damage after contraction‐induced muscle injury, and (3) neutrophils aid the resolution of contraction‐induced muscle injury. These hypotheses were tested by exposing extensor digitorum longus (EDL) muscles of mice deficient in CD18 (CD18−/−; Itgb2tm1Bay) and of wild type mice (C57BL/6) to in situ lengthening contractions and by quantifying markers of muscle inflammation, injury, oxidative damage and regeneration/repair. Neutrophil concentrations were significantly elevated in wild type mice at 6 h and 3 days post‐lengthening contractions; however, neutrophils remained at control levels at these time points in CD18−/− mice. These data indicate that CD18 is required for neutrophil accumulation after contraction‐induced muscle injury. Histological and functional (isometric force deficit) signs of muscle injury and total carbonyl content, a marker of oxidative damage, were significantly higher in wild type relative to CD18−/− mice 3 days after lengthening contractions. These data show that neutrophils exacerbate contraction‐induced muscle injury. After statistically controlling for differences in the force deficit at 3 days, wild type mice also demonstrated a higher force deficit at 7 days, a lower percentage of myofibres expressing embryonic myosin heavy chain at 3 and 7 days, and a smaller cross sectional area of central nucleated myofibres at 14 days relative to CD18−/− mice. These observations suggest that neutrophils impair the restoration of muscle structure and function after injury. In conclusion, neutrophil accumulation after contraction‐induced muscle injury is dependent on CD18. Furthermore, neutrophils appear to contribute to muscle injury and impair some of the events associated with the resolution of contraction‐induced muscle injury.

Exercise-induced muscle damage: effect on circulating leukocyte and lymphocyte subsets.

The purpose of this study was to determine the effect of downhill and level running on circulating leukocyte and lymphocyte subsets and T lymphocyte activation. Using a random cross-over design, 10 runners completed two trials of 60 min of level running (0% grade; LR) and downhill running (-10% grade; DHR) at 70% of level VO2max. Blood samples were obtained preexercise and immediately postexercise (POST) and at 1.5, 12, 24, and 48 h of recovery. Creatine kinase activity peaked at 12 h of recovery from DHR and was not significantly altered following LR. The number of total T, CD16+, CD3+CD56+ cells were significantly higher POST DHR compared with LR. Leukocyte and neutrophil counts were significantly higher at 1.5 and 12 h of recovery from DHR compared with LR. The number of activated CD8+ cells (CD25+ CD8+) was significantly higher at 12 h of DHR compared to LR. Total T cells were significantly reduced at various time points during the 48 h of recovery from LR and DHR. In summary, DHR relative to LR resulted in a greater mobilization of lymphocytes (post), neutrophils (1.5-12 h of recovery) and activation of CD8+ cells at 12 h of recovery. In addition, reductions in circulating T lymphocyte subsets occurred following both conditions.

Ibuprofen and Acetaminophen: Effect on Muscle Inflammation after Eccentric Exercise

PURPOSE We examined the influence of ibuprofen and acetaminophen on muscle neutrophil and macrophage concentrations after novel eccentric contractions. METHODS Twenty-four males (25 +/- 3 yr) were divided into three groups that received the maximal over-the-counter dose of either ibuprofen (1200 mg x d-1), acetaminophen (4000 mg x d-1), or a placebo after eccentric contractions of the knee extensors. Biopsies from the vastus lateralis were taken before and 24 h after exercise. Inflammatory cells were quantified in muscle cross-sections using immunohistochemistry. RESULTS Macrophage concentrations were elevated by 1.5- to 2.5-fold (P < 0.05) at 24 h postexercise relative to preexercise concentrations, whereas neutrophil concentrations were not significantly elevated. Muscle inflammatory cell concentrations were unaffected by treatment with ibuprofen or acetaminophen when compared with placebo. CONCLUSIONS Maximal over-the-counter doses of ibuprofen or acetaminophen, when administered therapeutically, do not affect muscle concentrations of neutrophils or macrophages 24 h after a novel bout of eccentric contractions.

The Effect of volume ingested on rehydration and gastric emptying following exercise-induced dehydration

The purpose of this study was to examine the effects of different drink volumes on rehydration, gastric emptying, and markers of fluid balance following exercise-induced dehydration. Nine male subjects (27.3 +/- 5.47 yr of age, 77.8 +/- 7.9 kg) exercised for 90 min (or until 2.5% of initial body weight was lost) on a cycle ergometer in a hot environment (30 degrees C with 60% RH). Following exercise, subjects were moved to a neutral environment (23 degrees C 50% RH) and rested for 30 min prior to beginning a 3-h rehydration period. During rehydration, subjects were serially fed with an electrolyte solution (14.98 mmol.l-1 Na+, 13.51 mmol.l-1 Cl-, and 7.95 mmol.l-1 K+) every 30 min with either 100% or 150% of the fluid lost during exercise. Gastric contents were determined every 15 min using double sampling. Blood samples, urine samples, and body weights were taken before and after exercise and at 1-h intervals throughout rehydration. Blood samples were analyzed for percent change in plasma volume, electrolyte concentration, aldosterone levels, and renin activity. Urine electrolyte concentrations were also measured. The final percent rehydration was 48.11 and 67.90 for the 100% and 150% conditions, respectively. During rehydration, the subjects emptied 98.9 and 86.0% of the fluid ingested, and the % emptied and used for weight gain at the end of rehydration was 55.1 and 54.6 for the 100% and 150% trials, respectively. Urine production was significantly higher in the 150 compared with the 100% condition while renin and aldosterone levels did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines derived from cultured skeletal muscle cells after mechanical strain promote neutrophil chemotaxis in vitro

We tested the hypothesis that cytokines derived from differentiated skeletal muscle cells in culture induce neutrophil chemotaxis after mechanical strain. Flexible-bottom plates with cultured human muscle cells attached were exposed to mechanical strain regimens (ST) of 0, 10, 30, 50, or 70 kPa of negative pressure. Conditioned media were tested for the ability to induce chemotaxis of human blood neutrophils in vitro and for a marker of muscle cell injury (lactate dehydrogenase). Conditioned media promoted neutrophil chemotaxis in a manner that was related both to the degree of strain and to the magnitude of muscle cell injury (ST 70 > ST 50 > ST 30). Protein profiling using a multiplex cytokine assay revealed that mechanical strain increased the presence of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, monocyte chemotactic protein (MCP)-1, and IL-6 in conditioned media. We also detected 14 other cytokines in conditioned media from control cultures that did not respond to mechanical strain. Neutralization of IL-8 and GM-CSF completely inhibited the chemotactic response for ST 30 and ST 50 and reduced the chemotactic response for ST 70 by 40% and 47%, respectively. Neutralization of MCP-1 or IL-6 did not reduce chemotaxis after ST 70. This study enhances our understanding of the immunobiology of skeletal muscle by revealing that skeletal muscle cell-derived IL-8 and GM-CSF promote neutrophil chemotaxis after injurious mechanical strain.

Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use.

The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water‐treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for l‐NAME‐treated animals compared with water‐treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between l‐NAME‐treated and control animals, suggesting that l‐NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells. J. Leukoc. Biol. 64: 427–433; 1998.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury.

UNLABELLED Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. STATEMENT OF SIGNIFICANCE COX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model. The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Sensory level electrical muscle stimulation: effect on markers of muscle injury

Background: Monophasic high voltage stimulation (MHVS) is widely prescribed for the treatment of inflammation associated with muscle injury. However, limited scientific evidence exists to support its purported benefits in humans. Objective: To examine the efficacy of early initiation of MHVS treatment after muscle injury. Methods: In a randomised, cross over design, 14 men performed repetitive eccentric contractions of the elbow flexor muscles followed by either MHVS or control treatment. MHVS treatments were applied five minutes and 3, 6, 24, 48, 72, 96, and 120 hours after eccentric contractions. Results: MHVS resulted in a significant reduction (p<0.05) in delayed onset muscle soreness 24 hours after eccentric exercise compared with controls. Elbow extension was significantly increased immediately after administration of MHVS compared with controls. No significant differences were observed between MHVS treatment and controls for maximal isometric strength, flexed arm angle, or arm volume. Conclusions: Early and frequent application of MHVS may provide transient relief from delayed onset muscle soreness and short term improvements in range of motion after injurious exercise. However, MHVS treatment may not enhance recovery after muscle injury because of lack of improvements in strength and active range of motion.

Run training versus cross-training: effect of increased training on circulating leukocyte subsets.

The purpose of this study was to determine the effects of increased training via cross-training (run + cycle) and run training on circulating leukocyte subsets. Male runners (N = 11) participated in two randomly assigned increased training (IT) periods after 30 d of normal training (NT). Each IT began after a 14 d period of reduced training (80% of NT) followed by 10 d of IT (200% of NT). During IT, the subjects ran in the afternoon for 10 d (100% NT) and performed 8 additional training sessions in the morning (100% NT) on a treadmill (ITRT) or a bicycle ergometer (ITCT). Blood samples were obtained before (D0), on day 5(D5) and after 10 d (D11) of ITRT and ITCT. A significant increase in the CD4+/CD8+ ratio occurred at D5 compared with D0 and D11. The CD4+/CD8+ ratio was significantly lower during ITRT compared with ITCT at D11. The number of circulating CD3+, CD4+, and CD8+ cells were significantly reduced at D11 compared with D0. In conclusion, 10 d of IT resulted in a significant reduction in the number of circulating T cells independent of the training mode and a reduction in the CD4+/CD8+ ratio for ITRT but not for ITCT.

Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.