This study assessed the time course of functional recovery of the quadriceps femoris muscle after injury induced by eccentric exercise at low angular speed. According to Warren et al., 1999, the measurement of muscle function provides the best means of evaluating the magnitude and time- course of muscle injuries resulting from eccentric contractions. Muscle function is operationally defined as the ability to exert force under a given set of conditions, that is, over a given range of motion or at a fixed muscle length, at a given velocity or at a given external load, at a given level of activation and over a given number of contractions. A tool that assesses one or more of these components of muscle function is defined as a functional measurement tool. MVC torque and electromyography were the tools used in the present study to assess muscle function. In addition, CK activity was determined and MRI carried out, in order to verify the effectiveness of the eccentric exercise protocol in inducing muscle injury. An increase in intensity of the MRI signal was shown for all parts of the quadriceps femoris muscle on the 2nd and 7th days after eccentric exercise, which remained high between the 21st and 30th days in two subjects. These results are in agreement with those of Shellock et al., 1991, who reported an increase in intensity of the signal for the elbow flexor muscles of 5 subjects after eccentric exercise. The authors found an increase in intensity of the signal during the days following eccentric exercise, which gradually decreased after the 10th day. Whilst in the present study alteration in intensity of the signal was used as the method, other studies have used the assessment of alterations in the T2 relaxation time as the method (Foley et al., 1999; Nosaka and Clarkson, 1996). It has been established that the T2 relaxation time increases during exercise, and then returns to the resting value within an hour post-exercise (Foley et al., 1999). However, Shellock et al., 1991 observed that a second increase in the T2 relaxation time developed gradually, giving a peak on the 3rd and 5th days after eccentric exercise, but not after concentric or isometric exercise. The time course and magnitude of this delayed increase in the T2 relaxation time, as also its relationship with other markers of muscle injury, have been described during the post-exercise period, with the general conclusion that the chronic T2 phenomenon reflects edema (Nosaka and Clarkson, 1996; Shellock et al., 1991). On the other hand, assessment of the increase in intensity of the signal after eccentric exercise, as identified by visual inspection, has been used in various previously published studies, being considered indicative of edema (Babul et al., 2003; Nurenberg et al., 1992; Shellock et al., 1991). According to Nurenberg et al., 1992, the immediate increase in intensity of the signal is a normal response occurring in parallel to the increase in intracellular and predominantly extracellular water that accompanies the exercise. To the contrary, the delayed rise in intensity of the signal seems to occur in parallel with delayed- onset muscle soreness and the ultra-structural injury, peaking from 48 to 96 hours after the exercise. These authors found high correlation between the increase in intensity of the signal assessed by MRI and the ultra-structural injury, as determined by an autopsy of some of the leg muscles (soleus, gastrocnemius lateralis, gastrocnemius medialis, anterior tibial and fibular longus) after carrying out the eccentric exercise. Based on a visual inspection, Nurenberg et al., 1992 used a scale from 0 to 5 (0 = normal, 5 = very severe signal intensity increase) to grade the increase in intensity of the signal after eccentric exercise. Similarly, Babul et al., 2003 recorded the intensity of edema using a subjective score based on visual assessment, where: 0 = no visible edema, 1 = minimal muscle edema, 2 = moderate muscle edema and 3 = marked muscle edema. In the present study, only the presence or absence of an increase in signal intensity (edema) was registered and the affected muscle, since it was not the objective of the present study to assess the degree of increase in signal intensity during the injury recovery period. Another important aspect refers to the MRI protocol used to assess the signal intensity. Part of the studies used T2-weighted images to assess the increase in signal intensity (Shellock et al., 1991). However, in the present study, after carrying out various pilot studies, assessment of the inversion-recovery sequence was chosen, described as preferable by some authors in identifying alterations in the amount of water observed in muscle injury (Fleckenstein et al., 1989). The prolonged decrease in strength after eccentric exercise is considered to be one of the most valid and reliable indirect measurements of muscle damage in humans (Warren et al., 1999). In the present study MVC torque decreased 56% immediately after eccentric exercise and remained low up to the 4th day. This result is similar to that observed by Clarkson et al., 1992. Clarkson et al., 1992 detected a 50% decrease in maximal isometric strength of the elbow flexor muscles immediately after maximal eccentric exercise. Although a decrease in muscle strength after eccentric exercise is a frequent finding, differences exist between studies when comparing the recovery time. For example, in the present study, the MVC torque decreased significantly up to the 4th day after eccentric exercise. However, in other studies a longer time was required to return to the pre-exercise strength levels (Bottas et al., 2005; Clarkson et al., 1992; Chen and Nosaka, 2006). For example, Clarkson et al., 1992 reported that up to the 10th day after eccentric exercise, the strength had still not returned to the pre-exercise level. Possible reasons for these differences in muscle strength recovery time could be differences between the injury induction models (Chen and Nosaka, 2006) or the functional role of the injured muscle or muscle group. Doubts still exist with respect to the mechanism involved in decreasing muscle strength after eccentric exercise (Clarkson and Hubal, 2002). According to McHugh, 2003, the loss in strength following a bout of eccentric exercise could theoretically be due to a physical disruption of the force-generating structures (including a loss of myofibrillar contractile proteins) or a failure to activate intact force-generating structures within the muscle fibre (excitation-contraction coupling). Impaired excitation-contraction coupling has been estimated to account for 50-75% of strength loss in the first 5 days following a damaging eccentric exercise (Warren et al., 2001). However, this estimate is based on electrically stimulated maximal contractions in an animal model, and little is known about the effects in human skeletal muscle with voluntary contractions. Jones, 1996 and Hill et al., 2001 reported that the manifestation of a decrease in muscle strength after eccentric exercise is the phenomenon known as low-frequency fatigue (LFF). There is a decreased capacity to produce strength with a low-frequency stimulus after exercise induced muscle damage, which can extend for one week after the exercise. Edwards et al., 1977 suggested that LFF occurred due to a failure in the excitation-contraction coupling process. Animal- conducted studies indicated a reduction in calcium released by the sarcoplasmic reticulum after exercise-induced muscle damage as the primary cause of LFF, offering evidence that muscle inability to produce maximal strength after eccentric exercise results from the impairment of excitation- contraction coupling processes (Byrd, 1992). Other studies also indicated failure in the excitation-contraction coupling process as a plausible cause of decrease in muscle strength after eccentric exercises in humans. Hill et al., 2001 reported that along with a 33% decrease in quadriceps femoris maximal voluntary torque, a significant decrease in torque production was observed with low-frequency stimulation, which presented significant correlation with the depression in calcium release. With respect to electrical activity, it was shown that the RMS of the VMO and RF muscles decreased significantly on the 2nd day after eccentric exercise, remaining low up to the 3rd day for the RF muscle. On the other hand there was no alteration in RMS of the VL muscle after eccentric exercise. The RMS results for the VMO and RF muscles immediately after and on the 1st day after the eccentric exercise were similar to those found by McHugh et al., 2000 and Bajaj et al., 2002, in which the authors reported no alterations in this period. However, a decrease in RMS was found in the present study for the VMO and RF muscles, on the 2nd day after eccentric exercise. According to Basmajian and De Luca, 1985, the RMS represents the number of activated motor units during contraction, and is generally used as a muscle activity measurement. At the same time, Fridén et al., 1983 studied myofibrillar injury and observed focal disorder in the striated band pattern in 32%, 52% and 12% of the muscle fibres, 1 hour, 3 days and 6 days, respectively, after intense eccentric exercise in humans. Thus these authors demonstrated that the proportion of severely injured muscle fibres was greater between the 1st and 3rd days after eccentric exercise. This period coincided with the decrease in RMS of the VMO and RF muscles, observed in the present study, and thus one can suggest that this decrease could have been due to a reduced number of activated muscle fibres during the isometric contraction. The MDF of the VMO muscle increased significantly immediately after eccentric exercise. On the other hand, the MDF of the RF muscle decreased significantly immediately after and on the 1st day after eccentric exercise. A significant decrease was also observed for the VL muscle on the 1st and 2nd days after eccentric exercise. MDF has been associated with the average speed of conduction of the muscle fibre action potential. Thus, a decrease in MDF during fatigue has been attributed to a decrease in conduction speed in activating muscle fibres (Arendt-Nielsen and Mills, 1985; Merletti et al., 2001). Motor unity recruitment with the increase in strength production progress from slow- oxidative motor units to fast-oxidative motor units and then to fast-glycolytic motor units (Henneman et al., 1965). However, there is evidence that during eccentric contraction, preferential recruitment occurs with respect to fast-glycolytic motor units (Nardone et al., 1989). Therefore, this preferred recruitment of fast-glycolytic motor units explains the greater susceptibility to fast muscle fibre damage. Linnamo et al., 2000 reported that a decrease in muscle fibre conduction speed might be related to selective damage of fast muscle fibres. Following selective damage of the fast fibres after eccentric exercise, slow fibres would be preferentially recruited. Since the conduction speed of the slow fibre action potential is lower, a decrease in MDF is to be expected. Thus, considering that the VL and RF muscles show greater amounts of fast fibres (Johnson et al., 1973; Staron et al., 2000), one could suggest that the selective injury of these fibres would explain the decrease in MDF after eccentric exercise. On the other hand, the different behaviour of the MDF of the VMO could be related to the higher proportion of slow fibres (Travnik et al., 1995) that this muscle possesses. As a function of the decreased proportion of fast fibres in comparison with the VL and RF muscles, it is possible that the eccentric exercise had a reduced influence on the VMO fibres, not causing a reduction in MDF. On the other hand, the increase in MDF of the VMO muscle could be related to the increase in firing rates of the activated motor units, with the objective of producing isometric force comparable with the pre-exercise values. Differently from the majority of studies using eccentric isokinetic exercise to induce muscle injury (Chen, 2003; Linnamo et al., 2000), the eccentric exercise of the present study used low angular speed. It has been speculated that fast motor units are recruited during high speed movements, whilst slow motor units are recruited during low speed movements (Ewing et al., 1990). Based on this speculation, one can assume that the induction of injury at different angular speeds would result in different behaviours of the MDF, since different types of fibre would be recruited. The influence of angular speed on the pattern of motor unit recruitment during isokinetic exercise was studied by Hutchins et al., 1998. However, these authors found no difference in the MDF of the quadriceps femoris muscle (vastus medialis and vastus lateralis), suggesting that the speed does not alter the motor unit recruitment pattern (no selective recruitment) during isokinetic exercise. The decrease in MDF of the VL and RF muscles encountered in the present study, allows for the suggestion that low angular speed eccentric exercise (5°/s) promotes a recruitment pattern of these muscles similar to that of the eccentric isokinetic exercise at higher speeds used by Linnamo et al., 2000 and Chen, 2003 (115°/s and 60°/s, respectively), since these authors also reported a decrease in MDF of the elbow flexor muscles after exercise. Serum CK activity was higher on the 2nd day after eccentric exercise, returning to the pre-exercise level on the 7th day after the proposed exercise. The time period during which higher serum CK activity occurred seems to be dependent on the type of exercise used to provoke muscle damage. The majority of studies that evaluated the CK level used maximal eccentric contraction or even downhill running, where sub-maximal eccentric contraction of the quadriceps femoris muscle occurs. Some studies (Byrnes et al., 1985; Eston et al., 1996) demonstrated that peak serum CK activity after downhill running occurred between the 1st and 2nd days, while other studies (Byrne et al., 2001; Clarkson et al., 1992; Lee and Clarkson, 2003; Schwane et al., 2000) observed the peak serum CK activity for maximal eccentric exercises between the 4th and 6th days after exercise. It was not the purpose of the present study verify during which day peak serum CK activity occurred. However, the increase observed on the 2nd day after the eccentric exercise was an indirect evidence of the occurrence of injury. Inter-subject variability is highlighted as one of the major problems with respect to the use of the CK activity measurement as a muscle damage indicator. Nosaka and Clarkson, 1996 noted a great variability in CK response amongst individuals submitted to the same eccentric exercise protocol. Accordingly, Fridén and Lieber, 2001 did not consider CK activity as a reliable measurement of the extent of muscle damage. These authors reported poor correlation between serum CK activity and ankle dorsiflexor muscle function (torque evoked by nervous stimulation) in rabbits on the 1st, 2nd, 7th, 14th and 28th day after eccentric exercise damage. The authors also reported that this result was not unexpected, since muscle fibre permeability to intramuscular enzymes may or may not be correlated to the cell contractile function. Although Fridén and Lieber, 2001 reported that the CK activity level was not correlated with the magnitude of muscle damage, they suggested that high levels of CK provided evidence of muscle damage in a binary manner - injured or not injured. Therefore, the high inter-subject variability with respect to the CK response may be the explanation of the great standard deviation observed for the measurements performed on the 2nd day after eccentric exercise in the present study. The level of circulating estrogen appears to be a factor influencing CK activity. Thus sex is described as a factor affecting rupture of the sarcolemma, and hence, the release of CK. Previous studies found lower levels for CK release in women than in men, both in the basal condition and during exercising to a degree comparable with working (Van der Meulen et al., 1991). This difference would be a result of the exposition of the musculoskeleton to estrogen, which appears to protect the muscle from CK release (Bär et al., 1990). Based on this idea, one could imagine that the CK activity would vary depending on the phase of the menstrual cycle during which injury was induced, since the level of estrogen circulating fluctuates. Recent studies, both in animals (Sotiriadou et al., 2003; 2006) and in humans (Thompson et al., 2006) offer support to the idea of estrogen influencing CK activity. Although there was no control in the present study of the phase of the menstrual cycle during which muscle injury was induced, the present authors believe this did not influence interpretation of the results, since correlating the intensity of the CK response with the magnitude of muscle injury was not an objective of the present study, aiming simply to use CK to confirm the presence of injury. Finally, based on the increase in signal intensity (2nd and 7th days after eccentric exercise) and on the increase in CK activity, one can conclude that the protocol of low angular speed eccentric exercise resulted in muscle injury, and can thus be used as a model to induce injury in future studies aiming to assess recovery time. |