Experimental Physiology (1992), 77, 757-760 Printed in Great Britain
THE PROTECTIVE EFFECT OF DAMAGING ECCENTRIC EXERCISE AGAINST REPEATED BOUTS OF EXERCISE IN THE MOUSE TIBIALIS ANTERIOR MUSCLE P. SACCO AND D. A. JONES Department of Physiology, University College and Middlesex Hospital School of Medicine, Gower Street, London WCIE 6BT (MANUSCRIPT RECEIVED 8 JUNE 1992, ACCEPTED 7 JULY 1992) SUMMARY
Using a damaging eccentric exercise regime of the mouse tibialis anterior (TA) muscle we have investigated the extent and time course of protection afforded by one bout of exercise against damage resulting from a second bout of activity. Maximal force and fibre morphology were preserved if the exercise was repeated within 21 days, but by 84 days muscles once again became susceptible to damage. Low-frequency force loss had a shorter time course of protection against repeated exercise, lasting less than 21 days. The results provide evidence for different mechanisms contributing to the development of muscle damage following eccentric exercise and provide a basis for characterizing the adaptive response of muscle to damaging exercise. INTRODUCTION
It is well known that exercise involving eccentric work (i.e. forced lengthening contractions) can result in delayed injury to muscle fibres, both in man and animals (Newham, Mills, Quigley & Edwards, 1983; McCully & Faulkner, 1985). Furthermore, the extent of muscle damage depends on whether the muscle has been exercised in this way in the recent past (Bymes, Clarkson, White, Hsieh, Frykman & Maughan 1985; Jones & Newham, 1985). The changes resulting from training which confer protection from subsequent damage are not known and the work of Newham, Jones & Clarkson (1987) suggests that an episode of eccentric exercise may protect against some aspects of muscle damage, but not others. To provide an experimental model in which it is possible to study the underlying mechanisms of this adaptation in a controlled manner we have examined the efficacy and time course of protection provided by eccentric exercise of the mouse tibialis anterior (TA) muscle on force loss and fibre damage after repeated exercise. METHODS The TA muscles of female mice (C57 B1/10 strain) aged 18-22 weeks were exercised using the technique described by Sacco, Jones, Dick & Vrbova' (1992). Mice were anaesthetized with 4-5 % (w/v) chloral hydrate I.P. (0-01 ml/g body wt). A lever arm attached to a rotary motor extended the foot and stretched the TA muscle from a flexed to an extended position whilst it was being stimulated via the peroneal nerve for 300 ms at 100 Hz using 100 ps square-wave pulses of supramaximal voltage. Stimulated lengthening contractions were repeated every 5 s for 10 min, after which the incision was sutured and the animal allowed to recover. Following exercise, animals were divided into four groups corresponding to recovery periods of 10, 21, 84 and 166 days before the same exercise protocol was repeated. Three days after the
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second exercise the force and contractile properties of exercised and contralateral control muscles were recorded in situ (Sacco et al. 1992). A control group of animals, examined 3 days after a single bout of exercise (corresponding to the period of maximal fibre necrosis), provided a comparison for the repeat exercise measurements. Immediately following force recordings mice were killed with a lethal injection of chloral hydrate and the TA muscles removed, weighed and snap frozen in liquid nitrogen for histology. Quantitative information on the degree of histological damage was obtained by counting the number, per microscope field, of 'damage foci' (defined as any fibre undergoing disintegration or mononuclear cell infiltration) at 200x magnification on cryostat sections stained with Haematoxylin and Eosin. All results are presented as means± S.E.M. RESULTS
Three days after exercise TA muscles had a maximal (100 Hz) force of 0*56±0-09 N which was equivalent to 52*3±4.9 % of the non-exercised control muscle force. This deficit was associated with a significantly reduced 40/100 Hz force ratio (0*41±0-04 compared with 0.61±0.01 for controls). Muscles examined 3 days after the first bout of exercise showed the typical appearance of eccentric exercise-induced damage (Fig. IA) with extensive invasion of the muscle by mononuclear cells, many of which were located within muscle fibres (6.1±1.4 damage foci/field being observed, Fig. 2A). Muscles allowed 10 days recovery between exercise bouts (Fig. lB) showed the presence of regenerating myotubes but an absence of newly damaged fibres. With a delay of 166 days between exercise bouts a mixture of larger fibres with central nuclei and new damage foci were seen which approached the single 3-day post-exercise value in number (Fig. lC). Force losses after the second exercise bout following 10 and 21 days recovery were significantly smaller than those of exercised muscles allowed 84 or 166 days recovery; the force decrements at these later stages were similar to those of muscles damaged for the first time (Fig. 2B). These results correlate well with quantitative assessments of histological damage (Fig. 2A) which showed significantly fewer damaged fibres in the 10- and 21-day recovery groups compared with the exercised control muscles. In contrast, a reduced 40/100 Hz force ratio was seen after exercise in all but the 10-day recovery muscles (Fig. 2C). DISCUSSION
Performing a single bout of eccentric exercise had a temporary protective effect against damage resulting from repeated exercise if this occurred within 21 days. Of the three indices of muscle damage evaluated, low-frequency force loss had the briefest duration of protection (less than 21 days), whereas both maximum tetanic force and fibre morphology were preserved against repeated exercise for between 21 and 84 days. This discrepancy in the time course of protection suggests that the characteristics of muscle damage may have different causal mechanisms. Thus prolonged low-frequency force loss may reflect damage to the contractile activation apparatus of the fibre (sarcoplasmic reticulum) as suggested by Jones (1981), whereas maximum force loss probably reflects the loss of contractile components through necrosis resulting from the inflammatory cell response. Human studies have shown that eccentric work leads to protection against further exercise-induced damage for at least 6 weeks after the first exercise bout (Bymes
759
PROTECTIVE EFFECT OF ECCENTRIC EXERCISE
tII
4 >' .I
Fig. 1. Histological appearance of mouse tibialis anterior muscle 3 days after eccentric exercise (A), and 3 days after a second bout of exercise following recovery periods of 10 days (B) and 166 days (C). Scale bar: 50 um.
-0
a
7A 6 r------------------------------------- ----------------_________ I 5I 4 3 1 / 2 0------
-V .~ ~ ~ ~ ~ 0- - -
*uo U.L
100 80 0-
60
.
_
~~~*
B
T
I
T -
-
-
L
40
20 0 70 < 060 _
.0-
C
0 50-
= 040 0
° 030 200 Log days after initial exercise
Fig. 2. Changes recorded 3 days after a second bout of exercise following different recovery periods. A, number of damage foci/microscope field; B, maximal tetanic force; C, 40/100 Hz force ratio. Each datapoint gives mean±S.E.M. of five to six muscles. Dashed lines represent mean values for muscles 3 days after a single bout of exercise; S.E.M., 1-4 (A), 4-9 (B) and 0-04 (C). * P < 0-02 (Student's t test) for differences compared with values obtained 3 days after a single exercise bout.
760
P. SACCO AND D. A. JONES
et al. 1985; Newham et al. 1987), and the shorter protection in the mouse is consistent with a greater metabolic turnover in this species. Schwane & Armstrong (1983) found that downhill running caused appreciable delayed-onset fibre necrosis in the vastus intermedius muscle of rats, but if preceded by a shorter training run 3 days earlier, then no damage ensued. However, the above study showed some variability in the degree of necrosis observed and no measurements of muscle contractile properties were made. Use of the mouse TA model allows greater control of the damaging exercise than the voluntary exercise regimes described for animals and man. The present findings characterize the duration of adaptation of both histological and functional indices of muscle damage resulting from eccentric exercise. The use of such an animal model may answer a number of important questions regarding the adaptive response of muscle to damaging exercise, such as whether the intensity of the initial training exercise relates to the degree of subsequent protection afforded to the muscle. We gratefully acknowledge the support of The Wellcome Trust. REFERENCES BYRNES, W. C., CLARKSON, P. M., WHITE, J. S., HSIEH, S. S., FRYKMAN, P. N. & MAUGHAN, R. J. (1985). Delayed onset muscle soreness following repeated bouts of downhill running. Journal ofApplied Physiology 59, 710-715. JONES, D. A. (1981). Muscle fatigue due to changes beyond the neuromuscular junction. In Human Muscle Fatigue: Physiological Mechanisms (Ciba Foundation Symposium 82), ed. Porter, R. & Whelan, J., pp. 178-196. Pitman Medical, London. JONES, D. A. & NEWHAM, D. J. (1985). The effect of training on human muscle pain and damage. Journal of Physiology 365, 76P. MCCULLY, K. K. & FAULKNER, J. A. (1985). Injury to skeletal muscle fibers of mice following lengthening contractions. Journal of Applied Physiology 59, 119-126. NEWHAM, D. J., MILLS, K. R., QUIGLEY, B. M. & EDWARDS, R. H. T. (1983). Pain and fatigue after concentric and eccentric contractions. Clinical Science 64, 55-62. NEWHAM, D. J., JONES, D. A. & CLARKSON, P. M. (1987). Repeated high-force eccentric exercise: effects on muscle pain and damage. Journal of Applied Physiology 63, 1381-1386. SACCO, P., JONES, D. A., DICK, J. R. T. & VRBOVA, G. (1992). Contractile properties and susceptibility to exercise induced damage of normal and mdx mouse tibialis anterior muscle. Clinical Science 82, 227236. SCHWANE, J. A. & ARMSTRONG, R. B. (1983). Effect of training on skeletal muscle injury from downhill running in rats. Journal ofApplied Physiology 55, 969-975.
THE PROTECTIVE EFFECT OF DAMAGING ECCENTRIC EXERCISE AGAINST REPEATED BOUTS OF EXERCISE IN THE MOUSE TIBIALIS ANTERIOR MUSCLE P. SACCO AND D. A. JONES Department of Physiology, University College and Middlesex Hospital School of Medicine, Gower Street, London WCIE 6BT (MANUSCRIPT RECEIVED 8 JUNE 1992, ACCEPTED 7 JULY 1992) SUMMARY
Using a damaging eccentric exercise regime of the mouse tibialis anterior (TA) muscle we have investigated the extent and time course of protection afforded by one bout of exercise against damage resulting from a second bout of activity. Maximal force and fibre morphology were preserved if the exercise was repeated within 21 days, but by 84 days muscles once again became susceptible to damage. Low-frequency force loss had a shorter time course of protection against repeated exercise, lasting less than 21 days. The results provide evidence for different mechanisms contributing to the development of muscle damage following eccentric exercise and provide a basis for characterizing the adaptive response of muscle to damaging exercise. INTRODUCTION
It is well known that exercise involving eccentric work (i.e. forced lengthening contractions) can result in delayed injury to muscle fibres, both in man and animals (Newham, Mills, Quigley & Edwards, 1983; McCully & Faulkner, 1985). Furthermore, the extent of muscle damage depends on whether the muscle has been exercised in this way in the recent past (Bymes, Clarkson, White, Hsieh, Frykman & Maughan 1985; Jones & Newham, 1985). The changes resulting from training which confer protection from subsequent damage are not known and the work of Newham, Jones & Clarkson (1987) suggests that an episode of eccentric exercise may protect against some aspects of muscle damage, but not others. To provide an experimental model in which it is possible to study the underlying mechanisms of this adaptation in a controlled manner we have examined the efficacy and time course of protection provided by eccentric exercise of the mouse tibialis anterior (TA) muscle on force loss and fibre damage after repeated exercise. METHODS The TA muscles of female mice (C57 B1/10 strain) aged 18-22 weeks were exercised using the technique described by Sacco, Jones, Dick & Vrbova' (1992). Mice were anaesthetized with 4-5 % (w/v) chloral hydrate I.P. (0-01 ml/g body wt). A lever arm attached to a rotary motor extended the foot and stretched the TA muscle from a flexed to an extended position whilst it was being stimulated via the peroneal nerve for 300 ms at 100 Hz using 100 ps square-wave pulses of supramaximal voltage. Stimulated lengthening contractions were repeated every 5 s for 10 min, after which the incision was sutured and the animal allowed to recover. Following exercise, animals were divided into four groups corresponding to recovery periods of 10, 21, 84 and 166 days before the same exercise protocol was repeated. Three days after the
758
P. SACCO AND D. A. JONES
second exercise the force and contractile properties of exercised and contralateral control muscles were recorded in situ (Sacco et al. 1992). A control group of animals, examined 3 days after a single bout of exercise (corresponding to the period of maximal fibre necrosis), provided a comparison for the repeat exercise measurements. Immediately following force recordings mice were killed with a lethal injection of chloral hydrate and the TA muscles removed, weighed and snap frozen in liquid nitrogen for histology. Quantitative information on the degree of histological damage was obtained by counting the number, per microscope field, of 'damage foci' (defined as any fibre undergoing disintegration or mononuclear cell infiltration) at 200x magnification on cryostat sections stained with Haematoxylin and Eosin. All results are presented as means± S.E.M. RESULTS
Three days after exercise TA muscles had a maximal (100 Hz) force of 0*56±0-09 N which was equivalent to 52*3±4.9 % of the non-exercised control muscle force. This deficit was associated with a significantly reduced 40/100 Hz force ratio (0*41±0-04 compared with 0.61±0.01 for controls). Muscles examined 3 days after the first bout of exercise showed the typical appearance of eccentric exercise-induced damage (Fig. IA) with extensive invasion of the muscle by mononuclear cells, many of which were located within muscle fibres (6.1±1.4 damage foci/field being observed, Fig. 2A). Muscles allowed 10 days recovery between exercise bouts (Fig. lB) showed the presence of regenerating myotubes but an absence of newly damaged fibres. With a delay of 166 days between exercise bouts a mixture of larger fibres with central nuclei and new damage foci were seen which approached the single 3-day post-exercise value in number (Fig. lC). Force losses after the second exercise bout following 10 and 21 days recovery were significantly smaller than those of exercised muscles allowed 84 or 166 days recovery; the force decrements at these later stages were similar to those of muscles damaged for the first time (Fig. 2B). These results correlate well with quantitative assessments of histological damage (Fig. 2A) which showed significantly fewer damaged fibres in the 10- and 21-day recovery groups compared with the exercised control muscles. In contrast, a reduced 40/100 Hz force ratio was seen after exercise in all but the 10-day recovery muscles (Fig. 2C). DISCUSSION
Performing a single bout of eccentric exercise had a temporary protective effect against damage resulting from repeated exercise if this occurred within 21 days. Of the three indices of muscle damage evaluated, low-frequency force loss had the briefest duration of protection (less than 21 days), whereas both maximum tetanic force and fibre morphology were preserved against repeated exercise for between 21 and 84 days. This discrepancy in the time course of protection suggests that the characteristics of muscle damage may have different causal mechanisms. Thus prolonged low-frequency force loss may reflect damage to the contractile activation apparatus of the fibre (sarcoplasmic reticulum) as suggested by Jones (1981), whereas maximum force loss probably reflects the loss of contractile components through necrosis resulting from the inflammatory cell response. Human studies have shown that eccentric work leads to protection against further exercise-induced damage for at least 6 weeks after the first exercise bout (Bymes
759
PROTECTIVE EFFECT OF ECCENTRIC EXERCISE
tII
4 >' .I
Fig. 1. Histological appearance of mouse tibialis anterior muscle 3 days after eccentric exercise (A), and 3 days after a second bout of exercise following recovery periods of 10 days (B) and 166 days (C). Scale bar: 50 um.
-0
a
7A 6 r------------------------------------- ----------------_________ I 5I 4 3 1 / 2 0------
-V .~ ~ ~ ~ ~ 0- - -
*uo U.L
100 80 0-
60
.
_
~~~*
B
T
I
T -
-
-
L
40
20 0 70 < 060 _
.0-
C
0 50-
= 040 0
° 030 200 Log days after initial exercise
Fig. 2. Changes recorded 3 days after a second bout of exercise following different recovery periods. A, number of damage foci/microscope field; B, maximal tetanic force; C, 40/100 Hz force ratio. Each datapoint gives mean±S.E.M. of five to six muscles. Dashed lines represent mean values for muscles 3 days after a single bout of exercise; S.E.M., 1-4 (A), 4-9 (B) and 0-04 (C). * P < 0-02 (Student's t test) for differences compared with values obtained 3 days after a single exercise bout.
760
P. SACCO AND D. A. JONES
et al. 1985; Newham et al. 1987), and the shorter protection in the mouse is consistent with a greater metabolic turnover in this species. Schwane & Armstrong (1983) found that downhill running caused appreciable delayed-onset fibre necrosis in the vastus intermedius muscle of rats, but if preceded by a shorter training run 3 days earlier, then no damage ensued. However, the above study showed some variability in the degree of necrosis observed and no measurements of muscle contractile properties were made. Use of the mouse TA model allows greater control of the damaging exercise than the voluntary exercise regimes described for animals and man. The present findings characterize the duration of adaptation of both histological and functional indices of muscle damage resulting from eccentric exercise. The use of such an animal model may answer a number of important questions regarding the adaptive response of muscle to damaging exercise, such as whether the intensity of the initial training exercise relates to the degree of subsequent protection afforded to the muscle. We gratefully acknowledge the support of The Wellcome Trust. REFERENCES BYRNES, W. C., CLARKSON, P. M., WHITE, J. S., HSIEH, S. S., FRYKMAN, P. N. & MAUGHAN, R. J. (1985). Delayed onset muscle soreness following repeated bouts of downhill running. Journal ofApplied Physiology 59, 710-715. JONES, D. A. (1981). Muscle fatigue due to changes beyond the neuromuscular junction. In Human Muscle Fatigue: Physiological Mechanisms (Ciba Foundation Symposium 82), ed. Porter, R. & Whelan, J., pp. 178-196. Pitman Medical, London. JONES, D. A. & NEWHAM, D. J. (1985). The effect of training on human muscle pain and damage. Journal of Physiology 365, 76P. MCCULLY, K. K. & FAULKNER, J. A. (1985). Injury to skeletal muscle fibers of mice following lengthening contractions. Journal of Applied Physiology 59, 119-126. NEWHAM, D. J., MILLS, K. R., QUIGLEY, B. M. & EDWARDS, R. H. T. (1983). Pain and fatigue after concentric and eccentric contractions. Clinical Science 64, 55-62. NEWHAM, D. J., JONES, D. A. & CLARKSON, P. M. (1987). Repeated high-force eccentric exercise: effects on muscle pain and damage. Journal of Applied Physiology 63, 1381-1386. SACCO, P., JONES, D. A., DICK, J. R. T. & VRBOVA, G. (1992). Contractile properties and susceptibility to exercise induced damage of normal and mdx mouse tibialis anterior muscle. Clinical Science 82, 227236. SCHWANE, J. A. & ARMSTRONG, R. B. (1983). Effect of training on skeletal muscle injury from downhill running in rats. Journal ofApplied Physiology 55, 969-975.
