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

Abstract

We tested the hypotheses that: (1) neutrophil accumulation after contraction-induced muscle injury is dependent on the beta(2) 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(-/-); Itgb2(tm1Bay)) 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.

Figure 1

A, neutrophil (Ly6G⁺ cells) concentrations, and B, percentage of fibres invaded by neutrophils after lengthening contractions. #Significantly higher for wild type relative to CD18−/− mice at the specified time point (interaction). *Significantly different from control levels (CT) for wild type mice only (time effect).

Figure 2

A, macrophage (F4/80⁺ cells) concentrations, and B, percentage of fibres invaded by macrophages after lengthening contractions. *Significantly different from control levels (CT) for both wild type and CD18−/− mice (time effect).

Figure 3. Force deficits

Force deficits calculated relative to values obtained prior to lengthening contractions, during recovery from lengthening contractions. #Significantly higher for wild type relative to CD18−/− mice at the specified time point (interaction).

Figure 4. Percentage of injured myofibres

#Significantly higher for wild type relative to CD18−/− mice at 3 days post-lengthening contractions (interaction). *Significantly different from control levels (CT) for both wild type and CD18−/− mice (time effect).

Figure 5

A, total carbonyl content. #Significantly higher for wild type relative to CD18−/− mice at 3 days post-lengthening contractions (interaction). *Significantly different from control levels (CT) for wild type mice only (time effect). B, carbonyl groups were found predominantly in the sarcoplasm of myofibres (arrow) (magnification 400 ×).

Figure 6

A, percentage of myofibres expressing eMHC. $Significantly higher for CD18−/− relative to wild type at 3 and 7 days post-lengthening contractions (group effect). B, localization of eMHC in a CD18−/− mouse 3 days after lengthening contractions. eMHC was found in normal sized myofibres (arrow), some of which had morphological characteristics of injury. Labelling was also found in apparent myotubes and/or immature myofibres (arrowhead) (magnification 600 ×).

Figure 7

A, percentage of central nucleated myofibres. *Significantly different from control levels (CT) for both wild type and CD18−/− mice (time effect). Cross sections of EDL muscles from wild type (B) and CD18−/− (C) collected 14 days after lengthening contractions that were stained with haematoxylin and eosin. Note the prevalence of central nucleated myofibres (arrows) (magnification 200 ×)).

Figure 8

A, CSA of central nucleated myofibres. #Significantly higher for CD18−/− relative to wild type at 14 days post-lengthening contractions (interaction) *Significantly different from 7 days post-lengthening contractions for both wild type and CD18−/− mice (time effect). B, frequency distribution of the CSA of central nucleated myofibres.