INTRODUCTION

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THE UNIVERSALLY EXPERIENCED phenomenon of delayed- onset muscle soreness (2) arises from damage to muscle fibers that is caused by their own contractions. The contraction-induced injury to muscle fibers results from a stretch or stretches of activated skeletal muscles, termed pliometric contraction(s) (11). The damage to muscle fibers that is induced by pliometric contractions occurs within single sarcomeres (6, 14, 18, 21, 23). During maximal isometric contractions of single fibers, heterogeneity in the lengths of sarcomeres develops along the length of the fiber (16, 17), particularly when fibers are activated at long lengths (8). The hypothesis is that relatively weak and strong sarcomeres exist in series with one another and that injury occurs within single weak sarcomeres when the thick and thin filaments are stretched beyond overlap, and structural elements within the sarcomere are damaged (18, 21, 23). The initial injury can be observed directly only by electron microscopy (EM; 6, 7, 11, 14, 23) or indirectly by measurement of a force deficit in the absence of fatigue (5, 6, 10, 18).

At 2-5 days after the initial injury to sarcomeres, the severity of the damage increases dramatically, whether measured by indirect measures of enzyme release (2, 10, 11), force deficit (5, 6, 11, 19, 20), or the subjective reports of pain by human beings (2, 10, 22) or by direct measures of the magnitude of the damage to fiber morphology by sections under light (10, 11, 20, 30) and electron microscopy (6, 11, 14, 18, 23). The increase in the morphological damage to fibers results from an inflammatory response, free radical damage, and phagocytosis of the sarcoplasm (10, 11, 31). In extensor digitorum longus (EDL) muscles of young mice (11, 20) and rats (30), the regeneration of new portions of the damaged muscle fibers (3) results in a complete recovery of muscle mass and maximal force within 14-30 days after the initial injury. Pliometric conditioning, with protocols that injure and allow regeneration of muscle fibers, provides protection from further injury in the muscles of small rodents (12) and of human beings (22). The newly regenerated portions of the fibers appear to be stronger and less susceptible to stretch and to injury (10, 12, 22).

Bupivacaine injection into EDL muscles of rats causes severe damage to muscle fibers within the first 2 days, and the maximal force developed by the muscle is decreased to ~10% of the control value (9, 24). The degeneration of muscle fibers is followed within 60 days by complete regeneration of muscle mass and recovery of ~90% of the maximal force. Our working hypothesis is that newly regenerated muscle fibers provide a population of sarcomeres that is more homogeneous, with respect to strength, than are the sarcomeres in mature fibers. Consequently, we designed an experiment in which EDL muscles of young (7 mo old) and old (26 mo old) male rats were exposed to a protocol of pliometric contractions known to injure EDL muscles in young and old animals to the same degree (11). We reasoned that bupivacaine treatment, along with the degeneration and regeneration of whole fibers, should provide a level of protection comparable to pliometric conditioning (12, 22). We tested the specific hypothesis that, 3 days after a protocol of pliometric contractions, the newly regenerated muscle fibers in bupivacaine-treated EDL muscles in both young and old rats would show a lesser deficit in maximal force and fewer damaged fibers than nontreated EDL muscles would show. Muscles of both young and old rats were studied because of the greater susceptibility of muscles in old animals to contraction-induced injury (5, 31) and the incomplete recovery after an injury in older animals (11).

	METHODS

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The initial experiments were carried out on the EDL muscles of 16 young (5 mo old) and 16 old (24 mo old) specific pathogen-free male Wistar-Car-Hicks rats. The rats were housed in the barrier facility of the Unit for Laboratory Animal Medicine at the University of Michigan. At the final evaluation, the young and old rats were 7 and 26 mo old, respectively. Walford (31) defined the beginning of old age arbitrarily as the age at which 50% of the cohort were deceased. The Wistar rats do not meet the criterion of 50% deceased until 28 mo of age, when they also show muscle atrophy and deficits in specific force (4, 9, 11). At 24 mo, the Wistar rats enter the steep slope of the survival curve; at 26 mo, 25% of their cohorts are deceased. Our designation of male Wistar rats 26 mo of age as "old" appears reasonable; for men, 25% of their cohorts are deceased at 65 yr of age (29), the traditional age of retirement. The present experiment was designed with the ages of the young and old rats selected so that no difference was likely to occur in the muscle mass or in maximal absolute or specific force of the EDL muscles. Under these circumstances, during each pliometric contraction, the average forces for muscles in young and old rats would not be different; consequently, the stimulus for the initial injury was the same for the two groups (5, 6, 18).

The young and old rats were each separated into two groups of equal number. In each age group, the left EDL muscle of each of eight rats was injected with 0.75% bupivacaine chloride (Marcaine; Sanofi, Winthrop, NY). Each muscle was administered as much bupivacaine as the muscle could hold, typically 125 µl, by means of three injections made along the length of the muscle: one injection each in the proximal, midbelly, and distal portions (9). Previous use of bupivacaine injections has indicated that by 2 days after the injection the development of maximal force is reduced to 10% of the value for the contralateral muscle and that by 60 days after the injection the recovery is to 85-90% (9). Sham injections of sterile saline injured 10-20 fibers at the site of the penetration of the needle (13). Because no evidence of damage was observed after the injection of the sterile saline, and recovery from the puncture wound was complete by 30 days (13), a sham-injected control protocol was unnecessary.

Measurement of maximal isometric tetanic force (P_o). The P_o of the left EDL muscle of each of the young and old rats in the treated and nontreated experimental groups was measured in situ immediately before (initial P_o) and 3 days after the administration of a pliometric contraction protocol (30). Rats were anesthetized with pentobarbital sodium (55 mg/kg ip), with supplementary doses administered to maintain a depth of anesthesia that prevented responses to tactile stimuli. Once a sufficient depth of anesthesia was reached, a small incision was made to expose the distal tendon of the EDL muscle. The intact distal tendon was clamped to the lever arm of a servomotor (Cambridge Technology). The servomotor served to displace the tendon of the muscle and to measure the force produced. The hindlimb was stabilized by pinning the knee and taping the foot to a platform that was heated with a thermal pad to maintain muscle temperature at ~35°C. Stainless steel needle electrodes were inserted on either side of the exposed peroneal nerve. The stimulation voltage and muscle length were adjusted to provide maximal isometric twitch force. This muscle length was defined as L_o. The fiber length (L_f) was determined by multiplication of L_o by a previously determined L_f /L_o ratio of 0.44 (4). A frequency-force curve was produced by stimulation of muscles at increasing frequencies from 10 to 180 Hz until the force plateaued at P_o.

Protocol of pliometric contractions. A protocol of 225 pliometric contractions (11, 20, 30) was administered to EDL muscles of young and old rats either 60 days after bupivacaine treatment or without bupivacaine treatment. This number of contractions was selected because the numbers of fibers damaged and the magnitude of the force deficits are not different for muscles of young and old mice (11). Contractions were initiated at L_o at a stimulation frequency of 100 Hz. For the first 350 ms of each contraction, the contraction was isometric; during the remaining period of the contraction, the muscle was stretched at a constant velocity of 1.5 L_f /s and then returned to starting length. The muscle was stretched through a strain of 20% relative to L_f once every 4 s. The protocol consisted of three 5-min bouts of 75 contractions, with 5 min of recovery between each bout. After the protocol, the incision was closed, and the rat was returned to a recovery cage.

Evaluation of damage to fibers. After the pliometric contraction protocol, the magnitude of the damage to the EDL muscles was evaluated indirectly by the measurement of a force deficit (6, 18-20, 30, 31) and directly by quantification of the number of damaged fibers in the muscle (10, 11, 20, 32). A force deficit was calculated as the difference between the initial P_o and the P_o measured 3 days after the protocol, expressed as a percentage of the initial P_o. After recovery from fatigue, the force deficit provides the most valid estimate of the totality of the damage (10).

Statistical analysis. Data are presented in the text and in Table 1 as means ± SE. Data were analyzed by a three-way analysis of variance, with randomized block design, by using the SAS statistical program (SAS Institute). On attainment of a significant F-statistic (P < 0.05), group means were compared by utilizing an alpha = 0.05 significance level and with the Bonferroni correction for multiple comparisons.

	RESULTS

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The nontreated and noninjured control EDL muscles in the leg contralateral to the experimental leg of young (7 mo) and old (26 mo) rats each developed a specific P_o (Table 1) not different from the values of ~240 kN/m² reported previously for adult mice (4) and rats (9, 31). Decreases in P_o and specific P_o of EDL muscles are not observed until mice (4) and rats (9) reach ~28 mo of age. At 60 days after the bupivacaine treatment, the treated EDL muscles had regenerated muscle masses and developed forces not different from the values for the nontreated muscles (Table 1). For nontreated EDL muscles of young and old rats, no age effect was evident 3 days after the pliometric contraction protocol, with force deficits of 62 and 72%, respectively. After recovery from bupivacaine treatment, the force deficits 3 days after the protocol were decreased dramatically, with values of ~18% for both young and old rats (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Force deficits (A) and %damaged fibers (B) in a single cross section of nontreated (solid bars; n = 16 rats) and bupivacaine-treated (open bars; n = 16 rats) extensor digitorum lungus (EDL) muscles in young (7 mo old; n = 16 rats) and old (26 mo old; n = 16 rats) rats 3 days after pliometric contraction protocol. Values are means ± SE.

Light microscopy of sections of nontreated muscles, fixed immediately after a pliometric contraction protocol known to produce a severe injury to nontreated muscles, showed no evidence of damaged fibers (Fig. 2B). In fact, these sections were not different from those of the uninjured control muscles (Fig. 2A). The EM of fibers from control EDL muscles (Fig. 3A) shows sarcomeres in alignment with an average sarcomere length of 2.65 ± 0.01 µm. This length for sarcomeres is in good agreement with the optimum sarcomere length for force development of 2.55 µm, normally cited for EDL muscles of rats (18).

View larger version (84K): [in this window] [in a new window]   Fig. 2.   Photomicrographs are of cross sections taken from EDL muscles of old rats. A: control EDL muscle; B: EDL muscle immediately after pliometric contraction protocol. Each section was stained with toluidine blue. Note that, when investigated at the light microscopic level, no damage to fibers was observed immediately after the protocol (B). Magnification, ×180.

View larger version (142K): [in this window] [in a new window]   Fig. 3.   Electron micrographs of longitudinal sections of a portion of a single fiber from uninjured control EDL muscle from an old rat (A) and from EDL muscle from old rat immediately after protocol with 225 pliometric contractions (B). B: arrow a indicates sarcomere with thick filaments displaced to 1 Z line; between the 2 b arrows, 7 myofilaments with sarcomeres show disruption of the thick- and thin-filament lattice; and 2 c arrows show "streaming" or discontinuities in Z line. Bars, 0.91 µm.

The EMs of single fibers from EDL muscles obtained immediately after the protocol showed focal areas of damage to sarcomeres (Fig. 3B). These EMs are not meant to be representative of the tens of millions of sarcomeres in a single fiber of an EDL muscle of a young or old rat. The EMs simply demonstrate the type of ultrastructural damage that occurs immediately after the pliometric contraction protocol (Fig. 3B) and provide evidence of immediate damage to sarcomeres. Immediately after the protocol, in the damaged area of a single fiber, most sarcomeres are severely out of alignment, and small groups of sarcomeres in series and in parallel meet the criteria of damage (see arrows, Fig. 3B). The sarcomeres identified as damaged were also stretched with a mean sarcomere length of 3.2 ± 0.11 µm, whereas intact sarcomeres in series with the damaged sarcomeres were shorter, with an average of 2.40 ± 0.05 µm. The mean length for intact sarcomeres in parallel with damaged sarcomeres of 2.61 ± 0.04 µm was not different from that of intact sarcomeres in control fibers. Sarcomere alignment and lengths were reestablished above and below the region of damaged sarcomeres (not shown). Although sampling is inevitably a problem with evaluations by EM, no apparent differences were observed in the type of injury to sarcomeres in muscles from young or old rats or from nontreated or bupivacaine-treated muscles. The difference was in the magnitude of the injury, as represented by the number of sarcomeres damaged.

In contrast to the lack of evidence of damage to fibers that was found by light microscopy immediately after the pliometric contraction protocol, 3 days after the pliometric contraction protocol, nontreated muscles (Fig. 4B) showed severe damage when compared with the uninjured control muscles (Fig. 4A). The decrease in the magnitude of the damage of treated compared with nontreated EDL muscles is evident qualitatively in the photomicrographs of cross sections from muscles obtained 3 days after the protocol (Fig. 4, C vs. B). An EM of a cross section of a single damaged fiber from a muscle 3 days after the protocol shows numerous phagocytes within an intact basement membrane and partial removal of the cytoplasm (Fig. 4D).

View larger version (138K): [in this window] [in a new window]   Fig. 4.   A: cross section from a control muscle taken from an old rat and stained with hematoxylin and eosin. B and C: EDL muscles removed from old rats 3 days after the protocol of 225 pliometric contractions. Morphological damage to fibers was most severe at this time. B: nontreated muscle; C: bupivacaine-treated muscle. Photomicrographs of comparable muscles from young rats and from old rats did not differ. D: electron micrograph of intact basement membrane (b arrows) of a fiber, with 1 part of the fiber devoid of myofibrils and the other part undergoing phagocytosis. Note numerous phagocytes ( p arrows). New portion of fiber regenerates within the old surviving basement membrane.

When evaluated quantitatively, the percentage of fibers damaged in the total cross section of nontreated EDL muscles was 46 and 50% for young and old rats, respectively. Consequently, our hypothesis that no age difference would exist regarding the protection afforded muscles by bupivacaine treatment could be tested rigorously without the problem of initial age differences in force deficit or numbers of fibers damaged. The hypothesis that bupivacaine treatment protects muscles of both young and old rats from contraction-induced injury was supported strongly, with the percentage of damaged fibers reduced from ~50 to ~14% for the bupivacaine-treated EDL muscles of both young and old rats (Fig. 1).

	DISCUSSION

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The protocol of 225 pliometric contractions has been administered to nontreated EDL muscles of both young (20) and old (11) mice and young rats (30). In the present study, the ~67% force deficit and ~48% damaged fibers for the nontreated EDL muscles of young and old rats were in excellent agreement with the values for force deficit and damaged fibers previously reported 3 days after the same protocol. Furthermore, the complete recovery of the muscle mass and force development of the bupivacaine-treated EDL muscles by 60 days after the protocol facilitated the subsequent comparisons with the nontreated muscles. For EDL muscles of both young and old rats treated with bupivacaine, the 75% decrease in the amount of damage, as measured by both the force deficit and the percentage of damaged fibers, provided dramatic evidence of the efficacy of the bupivacaine treatment in prevention of contraction-induced injury.

The mechanism by which the bupivacaine-treatment confers protection against contraction-induced injury appears to reside in the mechanical events responsible for the initial injury. Heterogeneity in the lengths of sarcomeres in series within myofibrils has been reported during maximal isometric contractions of single intact frog fibers, particularly when fibers are at lengths beyond L_o (8, 16, 17). The heterogeneity in the lengths of sarcomeres that develops during an isometric contraction appears to arise from the existence of regions of weaker sarcomeres in series with regions of stronger sarcomeres (1, 18, 21, 23). During maximal activation of single permeabilized fibers held at L_o, the stronger sarcomeres shorten or stay at the same length, whereas the weaker sarcomeres are stretched onto the descending limb of their length-force relationship (18). The sarcomeres stretched onto the descending limb are unstable and are at risk of being stretched excessively even during relatively small stretches of the overall fiber (18, 21, 23). Consequently, the phenomenon of heterogeneity in sarcomere length (8, 16, 17) plays a key role in the mechanical events that initiate contraction-induced injury (18, 21, 23).

The mechanism responsible for the initial injury to a maximally activated muscle by a single large stretch of >20% strain or by repeated small strains of <20% appears similar. In both cases, the initial injury occurs to single sarcomeres (10, 11); however, after the single stretch, the damage is more restricted and usually involves the displacement of some thick filaments to one Z line (7, 18). In the present experiments, disruption of the thick- and thin-filament lattice (18, 23) and "streaming" of the Z line (14, 23) were the more common forms of damage observed, with damage to substantial groups of sarcomeres. With increasing numbers of pliometric contractions, the initial injury within single sarcomeres is likely aggravated. The damage radiates throughout the sarcomere and to adjacent sarcomeres, both in series and in parallel (Fig. 3B). The increase in the damage with the number of pliometric contractions is supported by an attendant increase in the force deficit (19). The spread of the damage undoubtedly results from the manner in which the strain is transmitted serially within myofibrils and laterally throughout the fiber (26, 27). Inhomogenieties in the transmission of strain will place sarcomeres that bear greater strain at risk of injury and, once they are injured, the disruption of the homogeneous transmission of strain is lost completely by the excessive stretching of the damaged sarcomeres (18, 21).

During the days after the initial mechanical injury to single sarcomeres or groups of sarcomeres, the magnitude of the damage is aggravated significantly (11, 30, 31) by an inflammatory response, free radical damage, and eventual phagocytosis of the damaged portion of the fiber (see Fig. 4D). Regeneration of new portions of muscle fibers is initiated during the process of phagocytosis, when satellite cells are activated by an endogenous growth factor or factors (3). Satellite cells divide mitotically and form myotubes which differentiate and develop into newly regenerated portions in the mature muscle fibers (3, 25). For young mice (4, 11) and young rats (30), the recovery is complete within 14-28 days, whereas for old mice, after a severe injury with an ~60% force deficit, a 20% force deficit and a 20% loss of viable fibers remain even after 60 days (11). The muscles of old mice and rats do recover completely from less severe contraction-induced injuries (unpublished data, University of Michigan).

A number of investigations (13, 15) have demonstrated that the degeneration of fibers induced by the bupivacaine treatment results in the activation of satellite cells and a regenerative response that produces a population of young, newly regenerated fibers with nearly complete restoration of muscle mass and P_o (9, 24). We have now shown that these fibers that degenerate and regenerate are extremely resistant to contraction-induced injury. Similarly, a conditioning program that consists of repeated bouts of a pliometric contraction protocol, which initially injured fibers in the muscles of either small rodents (12) or human beings (22), ultimately produced a conditioned muscle in which the fibers were no longer injured. Our working hypothesis is that the resistance to contraction-induced injury of newly regenerated fibers, whether pliometrically conditioned or bupivacaine-treated muscles, resides in the greater homogeneity in the strength of the sarcomeres in series within the newly regenerated fibers. If injury is initiated within single sarcomeres through a "pulling apart" of the thick and thin filaments in weaker sarcomeres in series with stronger sarcomeres (18, 21, 23), a greater homogeneity in the strength of sarcomeres would provide protection from a stretch-induced injury. Through homogeneity in the strength of sarcomeres in series, a given stretch is shared equally among the sarcomeres in series. Consequently, either a larger stretch or a greater number of stretches are required to place some sarcomeres on the descending limb of the length-force relationship where sarcomeres are at risk of being pulled apart (18, 21, 23).

In contrast with the conditioning through a pliometric contraction protocol that initiates some degree of injury to nonconditioned muscles, neither isometric or miometric contraction protocols nor passive stretches injure muscles (20); consequently, they do not provide protection from the type of injury induced by pliometric contractions. Furthermore, the muscles of human beings appear to be more susceptible to injury than are those of small rodents, possibly because of humans' upright posture and the wide range of movements in occupational and leisure tasks that require pliometric contractions (10). For frail elderly human beings, the high degree of susceptibility to contraction-induced injury, the greater severity of the damage for a given insult, and the possibility of incomplete recovery (11) have grave consequences for individuals who are already compromised in their mobility and quality of life (28). These issues have major implications for the design of physical activity programs for frail elderly human beings. The muscles of the elderly require pliometric conditioning for protection from severe debilitating injuries (28), yet great caution is required to avoid inducing the very injury that the exercise program is designed to prevent (11).