Original Contributions
Free radical activity following contraction-induced injury to the extensor digitorum longus muscles of rats

https://doi.org/10.1016/S0891-5849(98)00317-7Get rights and content

Abstract

The purpose of the study was to investigate the role of free radicals in the injury induced by a protocol of repeated pliometric (lengthening) contractions to the extensor digitorum longus (EDL) muscle in situ in rats. Previous data have indicated that prior treatment with the antioxidant polyethylene glycol-superoxide dismutase reduced the damage that was apparent at 3 days following this type of exercise. Three hours and 3 days following the protocol, the magnitude of the semiquinone-derived free radical signal observed by electron spin resonance spectroscopy (ESR) was not different for exercised and non-exercised skeletal muscles. A reduction in the protein thiol content of muscle was evident at 3 h, and was still apparent at 3 days. Three hours after the protocol, the total muscle glutathione content and the percentage in the oxidized form were unchanged, but by 3 days the percentage of muscle glutathione present in the oxidized form was elevated. The susceptibility of muscle to lipid peroxidation in vitro was reduced 3 days after the pliometric contractions. These data indicate that oxidation of protein thiols and glutathione may be involved in the secondary damage following pliometric contractions, but provide no evidence that the species involved were derived from mitochondrial semiquinone radicals.

Introduction

Contraction-induced injury to skeletal muscle fibers is more likely to occur and is much more severe following pliometric (lengthening or eccentric) contractions than following miometric (shortening or concentric) contractions, isometric contractions or passive stretching of fibers [1]. The initial injury to muscle fibers appears to be mechanical and the magnitude of the damage is a function of the strain and the average force developed [2], [3], [4]. Protocols of single or multiple pliometric contractions produce an initial focal injury to single sarcomeres or small groups of sarcomeres within specific myofibres [5], [6]. The initial mechanical injury initiates a cascade of events that lead to a more severe late onset injury that peaks in rodents at approximately 3 d following the pliometric contractions [1]. There is some previous data indicating that the secondary injury in rodents is caused by increased production of free radical species. Administration of the antioxidant, polyethylene glycol-superoxide dismutase (PEG-SOD) protected mouse muscle fibers from the secondary injury and reduced the magnitude of the force deficit normally observed 3 d after a protocol of repeated pliometric contractions [7]. In contrast, supplementation of rats with the lipid soluble antioxidant, vitamin E, had no effect on the force deficit or the percentage of damaged fibres after a protocol of pliometric contractions [8], [9], although in our previous study, this intervention prevented the rise in serum creatine kinase activity [8].

Oxygen radical production during and after exercise has received considerable attention [10], but definite evidence for an involvement of oxygen radicals in exercise-induced muscle damage is lacking [11]. This appears to be due to the absence of specific techniques to reliably detect free radicals in complex biological tissues. Electron spin resonance is the only technique for direct analysis of free radical species and these techniques have shown an increase in a free radical signal in muscle, immediately following exhaustive exercise performed by rodents [12], [13]. In addition, increases in free radical activity have been demonstrated by indirect markers [14], [15], [16], [17]. However, these findings are not universal, for instance, Saxon et al. [18] studied the effects of either repetitive pliometric or miometric contractions of the knee extensors in man and found evidence of damaged muscle fibers following pliometric contractions, but neither protocol influenced indirect markers of free radical activity.

The purpose of the present investigation was to determine a number of indicators of free radical activity in a model system in that the contractile activity undertaken by a specific muscle could be precisely controlled. This was a rodent model comparable to that in that PEG-SOD was found to prevent the secondary injury [7]. Our hypothesis was that the protocol of pliometric contractions would cause an increase in the oxygen free radical content of muscles between 3 h and 3 d post-exercise (i.e., at the time when PEG-SOD prevented the secondary damage). Furthermore we hypothesised that the rise in oxygen free radical content would be demonstrable by both electron spin resonance analysis of muscle and serum and by measurement of secondary markers of free radical activity in muscle such as protein thiols, and oxidised glutathione. Finally we were also interested in whether the contractile activity would lead to a modification of the susceptibility of muscle tissue to oxidative stress in vitro.

Section snippets

Materials and methods

Measurements were made on 15 male specific pathogen free (SPF) Wistar rats with a mean age of 3 months. The rats were housed in barrier facilities of the Unit for Laboratory Animal Medicine at the University of Michigan. All animal procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals (US PHS, Pub. No. 85-23). One EDL muscle of each of 10 rats underwent the pliometric contraction protocol. The exercised rats were euthanized at 3 h (n = 5) or 3 d (n = 5)

Results

Typical free radical signals observed on electron spin resonance examination of EDL muscles at 77 K are shown in Fig. 1. These were characterised by their “g” value that was derived from the magnetic field strength and the microwave frequency at which the signal was seen. For all muscle samples a free radical signal of g value 2.0036–2.0040 was seen. At 3 h or 3 d after the protocol no differences in the amplitude of this signal were observed between EDL muscles that had been subjected to the

Discussion

Extensor digitorum longus muscles were studied at 3 h and 3 days postpliometric exercise because previous data indicate that at 3 h post-exercise, the initial post-exercise fatigue was effectively reversed, but by 3 d post-exercise, the secondary damage was maximal [8]. Furthermore, treatment of animals with PEG-SOD has previously been shown to prevent the secondary damage with no effect on the initial force deficit (at 3 h) [7]. We therefore tested the hypothesis that increased free radical

Acknowledgements

We would like to thank Dr. T. Taiwo for assistance with the ESR studies and the Wellcome Trust and the Physiological Society for travel grants (A.M.). The research performed at the University of Michigan was supported by The National Institutes on Aging Grant No. AG-06157.

References (37)

  • S.V Brooks et al.

    Injury to muscle fibres after single stretches of passive and maximally stimulated muscle in mice

    J. Physiol.

    (1995)
  • R.L Lieber et al.

    Muscle damage is not a function of muscle force, but active muscle strain

    J. Appl. Physiol.

    (1993)
  • J Friden et al.

    Myofibrillar damage following intense eccentric exercise in man

    Int. J. Sports Med.

    (1983)
  • S.C Macpherson et al.

    Contraction-induced injury to single fiber segments from fast and slow muscles of rats by single stretches

    Am. J. Physiol.

    (1996)
  • E Zerba et al.

    Free radical injury to skeletal muscles of young, adult and old mice

    Am. J. Physiol.

    (1990)
  • J van der Meulen et al.

    Contraction-induced injury to the extensor digitorum longus muscle of ratsrole of vitamin E

    J. Appl. Physiol.

    (1997)
  • J.A Warren et al.

    Elevated vitamin E does not attenuate exercise-induced muscle injury

    J. Appl. Physiol.

    (1992)
  • C.K Sen et al.

    Exercise and oxygen toxicity

    (1994)
  • Cited by (0)

    View full text