Response of macrophages in rat skeletal muscle after eccentric exercise

Abstract

Purpose: Macrophages are known to be important for healing numerous injured tissues depending on their functional phenotypes in response to different stimuli. The objective of this study was to reveal macrophage phenotypic changes involved in exercise-induced skeletal muscle injury and regeneration.

Methods: Adult male Sprague-Dawley rats experienced one session of downhill running (16° decline, 16 m/min) for 90 min. After exercise the blood and soleus muscles were collected at 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w after exercise, separately.

Results: It was showed that CD68⁺ M1 macrophages mainly infiltrated into muscle necrotic sites at 1-3 d, while CD163⁺ M2 macrophages were present in muscles from 0 h to 2 weeks after exercise. Using transmission electron microscopy, we observed activated satellite cells 1 d after exercise. Th1-associated transcripts of iNOS and Ccl2 were inhibited post exercise, while COX-2 mRNA was dramatically increased 12 h after running (p < 0.01). M2 phenotype marker Arg-1 increased 12 h and 3 d (p < 0.05, p < 0.01) after exercise, and Clec10a and Mrc2 were up-regulated in muscles 12 h following exercise (p < 0.05, p < 0.05).

Conclusion: The data demonstrate the dynamic patterns of macrophage phenotype in skeletal muscle upon eccentric exercise stimuli, and M1 and M2 phenotypes perform different functions during exercise-induced skeletal muscle injury and recovery.

Keywords: Eccentric exercise; Macrophage phenotype; Muscle injury; Regeneration.

Fig. 1

Serum Mb content of rats from all of the groups before and after one session of downhill running. *p < 0.05 vs. the control group. Mb: myoglobin. Values are plotted as mean ± SD.

Fig. 2

Representative immunohistochemical staining of M1 and M2 macrophages (brown color) in rat soleus section before and after one bout of downhill running, nuclei counterstained with hematoxylin (blue color). Scale bar was 20 μm.

Fig. 3

The amount of stained area (μm²) for M1 or M2 macrophages in rat soleus muscle sections before and after one session of downhill running. M1 macrophage: *p < 0.05, **p < 0.01 vs. 6 h after exercise; M2 macrophage: @p < 0.05 vs. the control group. Values are plotted as mean ± SD.

Fig. 4

Arg-1, Clec10a and Mrc-2 mRNA expression before and after one bout of downhill running in rat soleus muscles using real-time fluorescent quantitation PCR. Arg-1: *p < 0.05, **p < 0.01 vs. control group; Clec10a: ▲p < 0.05 vs. control group; Mrc-2: ★p < 0.05 vs. control group. Values are plotted as mean ± SD.

Fig. 5

COX-2, iNOS and Ccl2 mRNA expression before and after one bout of downhill running in rat soleus muscles using real-time fluorescent quantitation PCR. COX-2: @p < 0.01 vs. control group; iNOS: *p < 0.05, **p < 0.01 vs. control group; Ccl2: $p < 0.01 vs. control group. Values are plotted as mean ± SD.

Fig. 6

Satellite cells from soleus muscle were located between the membrane and the basal lamina surrounding the myofiber at 6 h and were activated 1 d after one session of downhill running, as shown in this transmission electron microscopy image. Scale bar: 11.0 × 10³ (at 6 h) or 4.80 × 10³ (at 1 d).

Fig. 7

Representative histological results for soleus muscles stained with hematoxylin and eosin before and after one session of downhill running in rats. Scale bar = 20 μm.