Myeloid Cell Responses to Contraction-induced Injury Differ in Muscles of Young and Old Mice

Abstract

Myeloid cells play a critical role in regulating muscle degeneration and regeneration. Thus, alterations with aging in the myeloid cell response to muscle damage may affect the progression of the injury in old animals. We hypothesized that neutrophil levels remain elevated and that macrophage accumulation is reduced or delayed in injured muscles of old compared with young animals. Muscles of young and old mice were injured with lengthening contractions and analyzed 2 or 5 days later. Regardless of age, neutrophil (Gr-1+) and macrophage (CD68+) content increased dramatically by Day 2. Between 2 and 5 days, macrophages increased further, whereas neutrophils declined to a level that in old muscles was not different from uninjured controls. M2 macrophages (CD163+) also increased between 2 and 5 days, reaching higher levels in muscles of old mice than in young mice. Although no evidence of persisting neutrophils or reduced M2 accumulation in old muscle was found, total macrophage accumulation was lower in old mice. Furthermore, messenger RNA levels showed age-related changes in macrophage-associated genes that may indicate alterations in myeloid cell function. Overall, differences between muscles of old and young mice in the inflammatory response through the early stages of injury may contribute to defects in muscle regeneration.

Figure 1.

Lengthening contractions damage muscles of young and old mice. The severity of injury was assessed 2 and 5 d after LCs by (A) the deficit in isometric force generating capacity expressed as a percentage of the contralateral control muscle, (B) the percentage of fibers in a single cross section showing histological evidence of damage, and (C) the total number of fibers appearing in a cross section. Data are presented as means ± SEM for muscles of young (black bars) and old (gray bars) mice. Two-factor ANOVA indicated significant effects of time and age without significant interactions for force deficit (Time: p = .008; Age: p = .004; Time × Age: p = .231) and number of injured fibers (Time: p < .001; Age: p = .008; Time × Age: p = .151) and significant effects of time with significant interactions for the number of fibers in cross sections with no main effect of age (Time: p < .001; Age: p = .319; Time × Age: p = .045). Sample sizes are n = 8–10 per group. Differences (p < .05) between individual groups are indicated by brackets. ANOVA = analysis of variance; LCs = lengthening contractions.

Figure 2.

Regeneration is impaired in old mice. Representative partial sections of muscles stained with hematoxylin and eosin from (A) young and (B) old mice show regenerating fibers. Scale bar = 50 µm and applies to both images. Data are shown for (C) the number of regenerating fibers per muscle cross section and (D) average cross-sectional area of regenerating fibers 5 d after lengthening contractions. Values are presented as means ± SEM for muscles of young (black bars) and old (gray bars) mice. Sample sizes are n = 8–10 per group. Differences (p < .05) between individual groups are indicated by brackets.

Figure 3.

Myeloid cell accumulation is altered in injured muscles from young and old mice. Data are shown for (A) neutrophil (Gr-1+ cells), (B) macrophage (CD68+ cells), and (C) M2 macrophage (CD163+ cells) content expressed as the number of cells per volume of muscle in uninjured control muscles and 2 and 5 d after LCs. Values are presented as means ± SEM for muscles of young (black bars) and old (gray bars) mice. Two-factor ANOVA indicated significant effects of time and age without significant interactions for neutrophil (Time: p < .001; Age: p < .001; Time × Age: p = .413) and M2 macrophage (Time: p = .039; Age: p < .001; Time × Age: p = .217) number and significant effects of time and age with significant interactions for the number of CD68+ macrophages (Time: p = .002; Age: p < .001; Time × Age: p < .001). Sample sizes are n = 8–10 per group. Differences (p < .05) between individual groups are indicated by brackets. *Differences within an age group from the control value. ^†Differences within an age group from the 2-d value. ANOVA = analysis of variance; LCs = lengthening contractions.

Figure 4.

Protein levels for myeloid cell markers increase following injury and with age. Data are shown for (A) Gr-1 (Ly6G and Ly6C), (B) CD68, and (C) CD163 protein levels in uninjured control muscles and 2 and 5 d after LCs determined by Western blot. Representative blots are shown in the right-hand panels. For analysis, protein levels were normalized to Ponceau S bands, which are also shown. Internal controls, randomly selected aged myotoxin-injected muscles, are included to allow densitometry measures to be compared between different blots. Left panels show average values expressed relative to the level in young control muscles and presented as means ± SEM for muscles of young (black bars) and old (gray bars) mice. Two-factor ANOVA indicated significant effects of time and age without significant interactions for CD68 levels (Time: p = .001; Age: p < .001; Time × Age: p = .377) and significant effects of time with strong trends for effects of age for Gr-1 (Time: p = .015; Age: p = .067; Time × Age: p = .398) and CD163 (Time: p < .001; Age: p = .078; Time × Age: p = .879) levels. Sample sizes are n = 4 per group. Differences (p < .05) between individual groups are indicated by brackets. *Differences within an age group from the control value. ^†Differences within an age group from the 2-d value. ANOVA = analysis of variance; LCs = lengthening contractions.

Figure 5.

Messenger RNA levels of M1 (iNOS, TNFα) and M2 (Arg1, IL-10) macrophage genes are altered with age. Shown are messenger RNA levels for iNOS (A, B), TNFα (C, D), Arg1 (E, F), and IL-10 (G, H) normalized to β2-microglobulin mRNA 2 d (A, C, E, G) and 5 d (B, D, F, H) after lengthening contractions for muscles of young (black bars) and old (gray bars) mice. Sample sizes are n = 8–10 per group. Differences (p < .05) between individual groups are indicated by brackets. Arg1 = arginase-1; IL-10 = interleukin-10; iNOS = inducible nitric oxide synthase; LC = lengthening contraction; TNFα = tumor necrosis factor alpha.

Figure 6.

Circulating levels of neutrophils and monocytes are elevated in old mice. Mean (A) neutrophil and (B) monocyte levels expressed as thousand cells per microliter blood ± SEM are shown for young (black bars) and old (gray bars) mice. Sample sizes are n = 6 per group. Differences (p < .05) between individual groups are indicated by brackets.