Body weight and leukocyte infiltration after an acute exercise-related muscle injury in ovariectomized mice treated with estrogen and progesterone.

In both rats and mice, an acute skeletal muscle injury leads to leukocyte infiltration in which the leukocytes remove dead fibers and cellular debris, induce a secondary injury, and/or promote myofiber regeneration. Short-term exogenous estrogen treatment attenuates this leukocyte infiltration and prevents body weight gain in rat exercise-induced skeletal muscle injury models. But these estrogen effects may not occur in mice because body weight gain does not consistently occur in ovariectomized mice treated with estrogen. Additionally, progesterone may also attenuate this leukocyte infiltration without affecting body weight. The aim of the current study was to compare body weight and leukocyte infiltration in exercise-injured skeletal muscle of ovariectomized mice treated with exogenous estrogen and progesterone for the short period of 17 days with that of ovariectomized-placebo-treated mice and gonadal-intact male and female mice. There was no significant difference in body weight between the ovariectomized-estrogen-treated and the ovariectomized-placebo-treated mice. The amount of intramuscular leukocyte infiltration of ovariectomized mice treated with estrogen or progesterone was not significantly different from that of ovariectomized-placebo-treated mice. However, in the injured muscle, the mean area of the leukocyte antigen, 7/4, of the ovariectomized-estrogen-treated group was 2-3-fold greater than that of the ovariectomized-placebo-treated, ovariectomized-estrogen-progesterone-treated, and intact male groups (p<.05), suggesting that the 7/4-positive leukocytes of the ovariectomized-estrogen-treated group were larger or had more antigen. In conclusion, ovariectomized mice demonstrate a different body weight and leukocyte response to short-term estrogen treatment than that of ovariectomized rats, and short-term estrogen treatment modulates leukocyte phenotype. These data broaden our understanding of estrogen's effects on body weight and leukocyte infiltration, and may aid in increasing our understanding of how males and females differ in response to acute muscle injury.

Macrophage colony stimulating factor-induced macrophage differentiation influences myotube elongation.

BACKGROUND: Unaccustomed exercise, high-intensity dynamic sports activities, or the resumption of normal weight-bearing after a period of disuse can induce skeletal muscle injury, which activates an inflammatory response followed by muscle regeneration. Specific subsets of macrophages are involved in muscle regeneration. But the exact role of macrophage differentiation during muscle regeneration remains to be elucidated. OBJECTIVE: The objective of the study was to examine the effect of macrophage colony stimulating factor (M-CSF)-differentiated, lipopolysaccharides (LPS)-stimulated-macrophage-conditioned medium on muscle-cell proliferation, fusion, and elongation, which are key events during muscle regeneration and myogenesis. METHOD: Murine C2C12 myoblasts were cultured in conditioned medium obtained from PU5-1R macrophages that were (a) undifferentiated, unstimulated; (b) M-CSF-differentiated, unstimulated; (c) undifferentiated, LPS-stimulated; or (d) M-CSF-differentiated, LPS-stimulated. Myoblast proliferation ratio, nuclei number, and length were measured. RESULTS: C2C12 cells cultured in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages had significantly more nuclei and greater length than cells cultured in conditioned medium from undifferentiated, LPS-stimulated macrophages. Dilution and denaturization of the M-CSF-differentiated, LPS-stimulated-macrophage medium prevented a marked increase in C2C12 nuclei number and length. However, the C2C12 myoblast proliferation ratio was significantly greater in conditioned medium from undifferentiated, LPS-stimulated macrophages than in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages. CONCLUSIONS: M-CSF-differentiated, LPS-stimulated macrophages may influence myogenesis and the early and terminal stages of muscle regeneration. This knowledge may aid in developing therapies that will directly expedite muscle repair and lead to faster rehabilitation and reduced rehabilitation costs.

The relation between immunohistochemical product discreteness and leukocyte manual quantification reliability.

The discreteness of immunohistochemical products may interfere with detecting individual cells when obtaining manual quantification even with the application of a nuclear stain. To examine the influence of discreteness, we: (1) determined the relation between manual quantification reliability and discreteness, (2) analyzed the reliability between the manual and computer-assisted approaches, and (3) determined the relation between the reliability of the manual and computer approaches and discreteness. Sixty murine injured skeletal muscle sections were immunolabeled using the antineutrophil antibody, 7/4. Using computer-generated section images, an observer counted the number of cells on 2 different occasions (T1 and T2). Computer cell counts per section and discreteness per section (as indicated by the max pixel area per section) were determined. A moderate correlation was found for the relation between manual reliability and discreteness (P<0.01). The rankings of manual and computer counts were different (P<0.01), suggesting a lack of reliability between these approaches. The relation between the reliability of the manual and computer approaches and discreteness was moderately correlated (P<0.01). Samples with a computer count 200 or more had a greater mean max pixel area than those with a count <200 (P<0.0001). In conclusion, manual quantification may be more appropriate with samples with a leukocyte count <200, because discreteness is higher within this cell count range. Alternatively, when samples consist of 200 or more cells and discreteness is low, then the approaches that account for discreteness should be utilized.