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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:527-533 (2006)
© 2006 The Gerontological Society of America

Protection From Contraction-Induced Injury Provided to Skeletal Muscles of Young and Old Mice by Passive Stretch Is Not Due to a Decrease in Initial Mechanical Damage

Nicole C. Lockhart and Susan V. Brooks

1 Department of Molecular and Integrative Physiology, 2 Department of Biomedical Engineering, and 3 Institute of Gerontology, University of Michigan, Ann Arbor.

Address correspondence to Susan V. Brooks, PhD, Institute of Gerontology, The University of Michigan, 300 N. Ingalls, Ann Arbor, Michigan 48109. E-mail: svbrooks{at}umich.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Contraction-induced injury occurs when muscles are stretched while activated (lengthening contractions). The injury is initiated by mechanical damage followed by an inflammatory response. Old animals are particularly susceptible to contraction-induced injury, yet exposure to stretches without activation (passive stretches) before lengthening contractions lessens the injury. We hypothesized that, for muscles of both young and old mice, prior exposure to passive stretches reduces the initial mechanical damage induced by lengthening contractions. Compared with unconditioned muscles in both age groups, administration of passive stretches 1 hour before lengthening contractions decreased the force deficit at 3 days by one half, but did not affect the force deficit at 10 minutes. Force deficits immediately after two lengthening contractions were also not different for passive stretch-conditioned and unconditioned muscles. The similarity in force deficits immediately following lengthening contractions for conditioned and unconditioned muscle indicates that passive stretch conditioning does not decrease initial mechanical damage in young or old mice.

SKELETAL muscles can be injured by their own contractions during everyday activities. Contraction-induced injury occurs most frequently during activities that involve a predominance of lengthening contractions in which a muscle is stretched while activated. Contraction-induced injury is initiated by the mechanical disruption of single or small groups of sarcomeres (1–3). The initial mechanical injury leads to a delayed secondary injury that includes an inflammatory response and the degeneration of injured fibers or portions of fibers (4,5). Aging increases susceptibility to contraction-induced muscle injury in rodents (6,7) and in human beings (8), and recovery from injury is impaired in old animals (9,10). The high susceptibility of muscles in old animals to injury is due at least in part to a greater initial injury as evidenced by larger isometric force deficits immediately following single lengthening contractions of whole muscles and single permeabilized fibers (6). Although the exact quantitative relationship between sarcomere disruption and the force deficit has not been determined, in the absence of fatigue, the isometric force deficit gives a reliable and reproducible measure of the initial mechanical damage (1,2,11).

Exercise training prior to exposure to lengthening contractions can reduce indices of damage (9,12–15). In humans, previous training with lengthening contractions reduced plasma creatine kinase levels, muscle soreness, and deficits in force following a subsequent bout of lengthening contractions (14). In rodents as well, prior exposure of a muscle to lengthening contractions significantly decreased the amount of morphological damage and the force deficit resulting from a subsequent bout of lengthening contractions (12,13,16). Although muscles of old animals can be conditioned for increased resistance to injury (12), prolonged structural and functional deficits observed in old animals following lengthening contractions (9,10) raise critical questions as to the risks involved in utilizing such conditioning programs in elderly people. As an alternative to conditioning with damaging lengthening contractions, stretches without activation (passive stretches) have also been shown to reduce contraction-induced injury in both young and old mice when administered prior to lengthening contractions (13,17).

Passive stretch conditioning may represent a safe and effective method of reducing contraction-induced injury in older individuals, but the mechanism by which passive stretch protects against injury is not known. Passive stretch conditioning could reduce the initial mechanical damage, the subsequent inflammatory response, or both. The initial mechanical damage would be reduced by changes that prevent the excessive stretch of sarcomeres (18). The muscle fiber cytoskeleton serves to stabilize sarcomeric structure, and several cytoskeletal proteins have demonstrated a sensitivity to stretch such that the stability of the cytoskeleton surrounding the sarcomeres may be increased (19–21). An increase in sarcomere stability would decrease the likelihood of large sarcomere strains and reduce the magnitude of the initial mechanical damage during contractions. Thus, our purpose was to investigate the effect of passive stretch conditioning on susceptibility to the initial mechanical damage in both adult and old animals. Our hypothesis was that, for muscles of both adult and old mice, prior exposure to a protocol of passive stretches would reduce the force deficit in the absence of fatigue immediately following a protocol of lengthening contractions.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
A total of 29 3- to 4-month-old specific pathogen–free (SPF) male C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). Old SPF male C57BL/6 mice were obtained at 24–25 months of age from the National Institute on Aging mouse colony at Harlan Sprague Dawley (Indianapolis, IN). Our sample of old mice originally totaled 17, but 4 mice died due to anesthesia overdose in the initial portion of the study. This problem was largely solved by more careful monitoring of the anesthesia in the old mice, and no additional mice were lost. All mice were housed in an SPF barrier facility in the Unit for Laboratory Animal Medicine at the University of Michigan until experimentation. Between experimental procedures, mice were housed in a separate SPF return room. All experimental procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan.

In Situ Evaluation of Contractile Properties
Each mouse was anesthetized by intraperitoneal injection of 2% avertin (.015 mL/g body weight). Supplemental doses (0.1 mL) were administered until the mouse failed to respond to a toe pinch. A small incision was made at the ankle and the distal tendon of the extensor digitorum longus (EDL) muscle was exposed. The mouse was placed on a platform that was warmed by circulating water maintained at 37°C. The hind limb was secured by pinning the knee with a blunt screw and tightly taping the foot to the platform. The intact tendon was then tied with 4.0 braided silk suture to the lever arm of a servomotor (Aurora Scientific, Richmond Hill, ON, Canada) which controlled the length of the muscle and measured the force generated. A computer controlled the servomotor and collected and stored force data. The small area of exposed tendon was kept moist by frequent administration of isotonic saline.

The EDL muscle was activated using an isolated stimulator (Grass Instruments, West Warwick, RI) and fine needle electrodes placed transcutaneously adjacent and parallel to the peroneal nerve. A pulse duration of 0.2 ms was used for all contractions. Twitch contractions were initiated at 6 V, and then increased in 1-V increments until a maximum force value was obtained, typically at about 10 V. Length of the muscle was then adjusted for maximum twitch tension. Optimal muscle length (Lo) was measured using well-defined anatomical landmarks as previously determined (9). Tetanic contractions were generated during trains of pulses with the stimulation frequency increased during successive trains in 50-Hz increments from 150 Hz until the force level plateaued at the maximum isometric force (Po). This technique minimized the number of activations required to determine Po, which was typically achieved at about 250 Hz. Between isometric contractions, the mouse was allowed to rest at least 1 minute with its knee unpinned to prevent blockage of blood flow and muscle fatigue. Optimal muscle fiber length (Lf) was determined by multiplying Lo by the previously determined Lf/Lo ratio of 0.44 (5).

In Situ Conditioning With Passive Stretches
Following evaluation of contractile properties, the EDL muscle was exposed to a conditioning protocol of stretches without activation (passive stretches). Seventy-five passive stretches were administered with a frequency of 0.25 Hz for a total duration of 5 minutes. Stretches were initiated at Lo and were of a 20% strain relative to Lf at a velocity of 1.0 Lf/s. Ten minutes after completion of the 75 passive stretches, Po was remeasured and mice remained anesthetized on the heated platform for 1 hour.

In Situ Lengthening Contraction Protocol and Evaluation
One hour following administration of the passive stretch-conditioning protocol, Po was within 90% of its initial value for both young and old mice. EDL muscles were administered a protocol of 75 lengthening contractions in situ (13). The protocol of 75 lengthening contractions was also administered to muscles in a separate group of mice without prior exposure to passive stretches (unconditioned group) to allow comparisons between conditioned and unconditioned muscles. Muscles in the unconditioned group underwent the same evaluation of contractile properties as those in the passive stretch-conditioned group followed by the immediate administration of the protocol of lengthening contractions. Lengthening contractions were identical to the passive stretches, except that stretches were initiated 100 ms after the onset of 150-Hz stimulation to allow the development of near Po prior to the stretch. Stimulation ceased at the end of the lengthening ramp. Lengthening contractions occurred at a frequency of 0.25 Hz for a total duration of 5 minutes.

In pilot experiments, Po was measured 10, 20, 30, and 40 minutes following lengthening contractions to monitor recovery from the fatigue present immediately following the protocol of 75 lengthening contractions. These pilot experiments showed, by two-way repeated-measures analysis of variance (ANOVA), that the initial force deficit of approximately 100% immediately following the lengthening contraction protocol decreased to about 60% within 10 minutes after lengthening contractions (Figure 1). No further decrease in force deficit was observed after 20, 30, or 40 minutes for muscles of either young or old mice, indicating that the portion of the force deficit due to fatigue was eliminated within 10 minutes and the remaining force deficit provided a measure of the magnitude of the initial injury (Figure 1). Consequently, for the remainder of the in situ experiments, the magnitude of the initial injury was estimated through measurements of Po 10 minutes after completion of the lengthening contractions. After the force measurement at 10 minutes, the small incision at the ankle was closed with 7.0 sterile monofilament nylon suture and bathed with povidone–iodine solution, and the mice were monitored until they recovered from anesthesia.


Figure 01
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  		Figure 1. Force deficits of extensor digitorum longus muscles 0, 10, 20, 30, and 40 minutes following administration of 75 in situ lengthening contractions for both young (3–4 months, n = 4) and old (24–25 months, n = 4) mice. Force deficit was calculated as the percent decrease in maximum isometric force produced before and after the bout of lengthening contractions. Values are means with standard error bars. No significant effect of age was observed, and force deficits 10, 20, 30, and 40 minutes after lengthening contractions were not different from one another

 
To assess whether passive stretches administered only 1 hour prior to lengthening contractions elicited protective adaptations in old animals as previously demonstrated for a period of 14 days between administration of passive stretches and lengthening contractions (17), mice were re-anesthetized after 3 days, and contractile properties were evaluated in situ as described above. Force deficit was defined as the difference between the Po measured immediately prior to lengthening contractions and the Po measured 3 days after lengthening contractions (expressed as a percentage of the pre-injury Po).

Histological Evaluation
After the final in situ evaluation, both EDL muscles were removed and deeply anesthetized mice were killed by the creation of a pneumothorax. EDL muscles were then trimmed of their tendons, weighed, coated in tissue freezing medium, and frozen in isopentane cooled by dry ice. Muscles were sectioned through the mid-belly at 10 µm using a cryostat and stained with hematoxylin and eosin. A single entire cross-section from each muscle was used to determine the number of degenerating and regenerating fibers as well as the total number of fibers per section. Sections were viewed with the aid of a microscope imaging system (Bioquant, Nashville, TN) which tallied the fiber counts. Degenerating fibers included those with pale or variable staining, clear infiltration of inflammatory cells, or a considerably swollen appearance (13). Regenerating fibers were defined as having nonperipheral nuclei without other evidence of degeneration. The numbers of degenerating and regenerating fibers were summed and reported as a percentage of the total fiber number for the section.

In Vitro Evaluation of Contractile Properties
In a second set of experiments, susceptibility to initial mechanical damage was assessed in vitro. The in vitro protocol was patterned after protocols used previously to distinguish muscles of adult and old animals (6), as well as dystrophic and control muscles (22,23), on the basis of susceptibility to initial mechanical damage in the absence of fatigue. Mice were anesthetized with intraperitoneal injections of avertin as described above. The EDL muscles were isolated, and 5.0 silk suture tied securely to the distal and proximal tendons. Muscles were carefully removed from the animal and placed in a horizontal bath containing buffered mammalian Ringer solution (composition in mM: 137 NaCl, 24 NaHCO3, 11 glucose, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, and 0.025 tubocurarine chloride) maintained at 25°C by a circulating water bath and bubbled with 95% O2–5% CO2 to stabilize pH at 7.4. One tendon of the muscle was tied to a force transducer (model BG-50; Kulite Semiconductor Products, Leonia, NJ) and the other tendon to the lever arm of a servomotor (model 305B; Aurora Scientific). After removal of the muscles, deeply anesthetized animals were euthanized by creation of a pneumothorax. Muscles were stimulated between two stainless steel plate electrodes with a square pulse of duration 0.2 ms. Current was increased from 100 mA until a maximum twitch tension was reached, typically between 300 and 400 mA. Length of the muscle was then adjusted for maximum twitch tension. Tetanic contractions were generated during 300-ms trains of pulses with the stimulation frequency increased during successive trains in 50-Hz increments from 150 Hz until the force level plateaued at the Po. Lo was measured with digital calipers.

In Vitro Conditioning and Injury
Following completion of the evaluation of contractile properties, half of the muscles underwent a passive stretch-conditioning protocol identical to that administered in situ. After 75 passive stretches were administered over a 5-minute duration, conditioned muscles remained undisturbed in the temperature-controlled bath for 1 hour with 95% O2–5% CO2 bubbling. After 1 hour, Po was remeasured and was within 90% of its initial value for both young and old mice. A protocol of two lengthening contractions separated by 10 seconds was administered to the conditioned muscles (6,22,23). An identical protocol of two lengthening contractions was also administered to muscles not previously exposed to passive stretches. The two lengthening contractions were each of 40% strain relative to Lf at a velocity of 1.0 Lf/s. The usefulness of any contraction protocol for discriminating between muscles for susceptibility to injury requires that the protocol be neither too benign nor too rigorous. For protocols that are too benign, neither conditioned nor unconditioned muscles would be significantly affected, whereas protocols that are too rigorous may overwhelm any differences due to passive stretch conditioning. The strain of 40% used in the present experiment was chosen to achieve a large enough force deficit for detecting a possible protective effect of passive stretch conditioning based on previous work indicating that stretches of smaller strains resulted in only minimal force deficits in unconditioned muscles (6,22,23). Stretches were initiated at Lo 100 ms after the onset of 150-Hz stimulation to allow the development of near Po prior to the stretch. Stimulation ceased at the end of the lengthening ramp. Po was measured 1 minute following the second lengthening contraction. Force deficits were calculated as the decrease in isometric force from just prior to the lengthening contractions to 1 minute after the second lengthening contraction and were expressed as a percentage of the Po prior to the stretch. Following completion of all force measurements, the muscle was removed from the bath, trimmed of tendon, and weighed.

Data Analysis
Physiological cross-sectional area was calculated by dividing wet muscle mass by the product of Lf and the muscle density constant, 1.06 mg/mm3. Specific Po was calculated by dividing Po by the physiological cross-sectional area. Means and standard errors were calculated for all variables. The body masses, muscle masses, and contractile properties for young and old mice were compared by using Student's t test. The force deficits and percentages of injured fibers were compared for unconditioned and passive stretch-conditioned muscles of both young and old mice using two-way ANOVA. In circumstances when the F ratio was statistically significant, Tukey multiple comparison tests were used to assess differences between individual means (SigmaStat; Sigma Chemical, St. Louis, MO). Statistical significance was set at p <.05.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Although the old mice used in this study had slightly larger body masses than the young mice, the mean values of EDL muscle mass for young and old mice were not different (Table 1). Despite no difference in muscle masses, the mean values for Lo, Po, and specific Po were all significantly lower for the EDL muscles of old compared with young mice (Table 1).


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  		Table 1. Descriptive Properties of the Experimental Sample.

 
Passive Stretch Protects Against Contraction-Induced Injury in Both Young and Old Mice
In young mice, administration of passive stretches 1 hour prior to in situ lengthening contractions significantly reduced the force deficit at 3 days from 50.7 ± 3.6% for unconditioned muscles to 16.7 ± 4.0% for passive stretch-conditioned muscles (Figure 2). Administration of passive stretches 1 hour prior to lengthening contractions also reduced the force deficit at 3 days for old mice, from 61.7 ± 8.5% in unconditioned muscles to 21.6 ± 8.9% in passive stretch-conditioned muscles. Similar to the data on force deficits, the percentage of fibers that demonstrated morphological evidence of injury 3 days following lengthening contractions was 20.5 ± 3.2% for unconditioned muscles of young mice compared with only 11.7 ± 1.5% for passive stretch-conditioned muscles. Prior conditioning with passive stretches also decreased the percentage of injured fibers in muscles of old mice from 26.3 ± 2.7% to 16.1 ± 1.2% (Figure 3). No effect of age was found for either the force deficit (p =.17) or the percentage of injured fibers (p =.08) 3 days following lengthening contractions, but the power for detecting an age effect was low for both of these tests (0.16 and 0.30, respectively), suggesting that the sample size, particularly of old mice, was too small to detect an increase in susceptibility to injury with age. No significant interactions were found between age and conditioning for either force deficit or injured fibers at 3 days. These data demonstrate that, consistent with previous reports of protection provided by passive stretch conditioning in young and old mice when passive stretches and lengthening contractions were separated by 14 days (17), the present conditioning protocol resulted in similar protective effects in both age groups.


Figure 02
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  		Figure 2. Force deficits of extensor digitorum longus muscles 3 days following a single bout of 75 in situ lengthening contractions in young (3–4 months) and old (24–25 months) mice that were unconditioned (young, n = 10; old, n = 4) or conditioned with a single bout of 75 passive stretches 1 hour prior to administration of the lengthening contraction protocol (young, n = 8; old, n = 4). Force deficit was calculated as percent decrease in maximum isometric force produced before and after the bout of lengthening contractions. Values are means with standard error bars. *Significant difference from unconditioned group (p <.05). No effect of age was observed

 

Figure 03
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  		Figure 3. Percentages of injured fibers in cross-sections of extensor digitorum longus muscles 3 days following a single bout of 75 in situ lengthening contractions in young (3–4 month) and old (24–25 month) mice that were either unconditioned (young, n = 7; old, n = 4) or conditioned with a single bout of 75 passive stretches 1 hour prior to administration of the lengthening contraction protocol (young, n = 7; old, n = 4). Percentages of injured fibers were calculated as the percentage of the total number of fibers in a cross-section that demonstrated clear signs of degeneration or regeneration. Values are means with standard error bars. *Significant difference from unconditioned group (p <.05). No effect of age was observed

 
Passive Stretch Does Not Reduce the Initial Mechanical Injury for Either Young or Old Mice
Based on our observation of no differences between force deficits measured at 10, 20, 30, and 40 minutes following in situ lengthening contractions (Figure 1), the force deficit at 10 minutes was chosen as an index of initial damage to minimize the length of time the mice had to be anesthetized. No effect of age or conditioning was found for the force deficits measured in situ at 10 minutes (Figure 4). The force deficits for unconditioned and passive stretch-conditioned muscles were 49.3 ± 4.6% and 48.2 ± 3.9%, respectively, for young mice and 58.4 ± 8.0% and 62.4 ± 4.8% for old mice. Although the effect of age on the force deficit 10 minutes following lengthening contractions did not reach statistical significance (p =.055), the power of this test (0.38) also indicated that our sample sizes were too low to detect an age effect.


Figure 04
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  		Figure 4. Force deficits of extensor digitorum longus muscles 10 minutes following a single bout of 75 in situ lengthening contractions in young (3–4 month) and old (24–25 month) mice that were unconditioned (young, n = 10; old, n = 4) or conditioned with a single bout of 75 passive stretches 1 hour prior to administration of the lengthening contraction protocol (young, n = 8; old, n = 4). Force deficit was calculated as percent decrease in maximum isometric force produced before and after the bout of lengthening contractions. Values are means with standard error bars. No significant effect of either age or conditioning was observed

 
Prior conditioning with passive stretches had no effect for either age group on the force deficits measured 1 minute after two 40% stretches in vitro (Figure 5). Although the force deficits were not different for age-matched passive stretch-conditioned and unconditioned muscles, consistent with previous reports of greater susceptibility of muscles in old animals to the initial mechanical damage induced by lengthening contractions (6) the force deficits of 27.4 ± 1.9% and 28.2 ± 0.8% for conditioned and unconditioned muscles, respectively, of old mice were significantly larger than the force deficits for conditioned (21.5 ± 2.4%) and unconditioned (20.8 ± 1.0%) muscles of young mice (Figure 5).


Figure 05
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  		Figure 5. Force deficits of extensor digitorum longus muscles 1 minute following two 40% in vitro lengthening contractions in young (3–4 month) and old (24–25 month) mice that were unconditioned (young, n = 6; old, n = 4) or conditioned with a single bout of 75 passive stretches 1 hour prior to administration of the lengthening contraction protocol (young, n = 6; old, n = 4). Force deficit was calculated as percent decrease in maximum isometric force produced before and after the bout of lengthening contractions. Values are means with standard error bars. *Significant difference from corresponding young group (p <.05). No significant effect of conditioning was observed

 

   DISCUSSION
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Our present observation of a reduction in force deficit and number of injured fibers 3 days following lengthening contractions for muscles exposed to conditioning with passive stretches only 1 hour prior to lengthening contractions is consistent with the previous demonstration of protection from contraction-induced injury when passive stretches and lengthening contractions were separated by 2 weeks (17). The protocol of 75 lengthening contractions used in the present study produced a force deficit at 3 days for unconditioned muscles of old mice of ~60%, as opposed to the protocol used in the previous study of 225 lengthening contractions that resulted in a force deficit of ~80% (17). Despite the reduced severity of the lengthening contraction protocol used in the present study, conditioning with passive stretches still reduced both the force deficit and the percentage of injured fibers in old mice. Furthermore, similar responses to passive stretch conditioning were observed for muscles of young and old mice, with passive stretch conditioning decreasing (for both age groups) the force deficit by nearly two thirds and the number of injured fibers by close to one half compared with those in unconditioned muscles. Unlike some previous studies (12,16), ours found no evidence of any impairment, compared with muscles of younger animals, in the ability of the muscles of old mice to respond to exercise conditioning.

Contrary to our hypothesis, force deficits soon after lengthening contractions did not differ between unconditioned and passive stretch-conditioned muscles when lengthening contractions were administered either in situ or in vitro. The observation that a protocol of passive stretches that is known to lessen contraction-induced injury did not diminish the force deficit measured 10 minutes following in situ lengthening contractions indicates that a reduction in mechanical injury is not responsible for the observed protection. The present result is consistent with previous findings that passive stretches administered either once immediately prior to lengthening contractions (24) or every other day for 12 days prior to lengthening contractions (25) did not decrease the force deficit immediately following the lengthening contractions, although neither of these studies investigated whether the passive stretch regimen used was sufficient to reduce contraction-induced injury 3 days following lengthening contractions. Further evidence that passive stretch does not reduce mechanical damage was provided by the similar force deficits observed in the present study for unconditioned and passive stretch-conditioned muscles following two lengthening contractions in vitro. The use of the two lengthening contractions in vitro, an established model of inducing damage in the absence of fatigue (6,22,23), is particularly important because the effect of age on muscular fatigue is disputed (26). Although the results of the in vitro experiments were consistent with the findings in situ of no protection from mechanical damage provided by passive stretches, we cannot discount the possibility that the lack of an effect of passive stretch to reduce the force deficit measured following lengthening contractions in vitro is due to the necessity of circulating or other factors not present in the bath to invoke the protective adaptation.

Our observations of a larger force deficit for muscles of old compared with young mice immediately following two 40% stretches in vitro and a trend for larger force deficits (p =.055) for old animals 10 minutes after 75 20% stretches in situ were consistent with previous findings of greater initial mechanical damage in muscles of old animals (6,7). The exact mechanism for increased initial injury in old animals is unknown, but the strong correlation between sarcomere length heterogeneity and the magnitude of mechanical damage (18,27) supports the hypothesis of increased sarcomere heterogeneity within aged muscles. Although the magnitude of initial injury differs between the age groups, the comparable responses to passive stretch conditioning that were observed in muscles of young and old animals may imply that passive stretches lead to similar protective adaptations in young and old mice, presumably via a decrease in secondary injury. Whereas we did not observe a significant difference in force deficit between young and old animals at 3 days, a previous study observed larger force deficits in old mice than in young mice both 10 minutes and 3 days following lengthening contractions (7). The difference in force deficit between young and old mice 3 days following lengthening contractions observed in the study by Zerba and colleagues (7) could be attributed to their use of somewhat older mice (26–27 months). In addition, the force deficits observed by Zerba and colleagues for the muscles of the young mice were smaller (42.3 ± 3.0%) than those observed in the present study (50.7 ± 3.6%). There is also the possibility that the in situ portion of the present study was underpowered due to small sample sizes, particularly for old mice 3 days following lengthening contractions.

Although passive stretches reduce contraction-induced injury when administered as little as 1 hour or as much as 14 days prior to lengthening contractions (13,17, present study), the mechanisms responsible for protection at these two time points might be very different. In particular, longer periods of separation between the conditioning and injury protocols allow for the feasibility of additional mechanisms that could not be invoked within 1 hour, such as increased transcription or translation. For passive stretch to provide protection against the initial mechanical damage, the adaptive changes have to be in place prior to administration of the lengthening contractions. Passive stretch could reduce mechanical damage by increasing the levels of proteins that stabilize cytoskeletal structures surrounding the sarcomeres, such as desmin, dystrophin, talin, or vinculin. Each of these cytoskeletal proteins demonstrates a sensitivity to stretch resulting in alterations in protein levels and/or properties (19–21). Although a mechanism requiring increased transcription or translation of new cytoskeletal proteins would not be feasible for the present study, where conditioning and injury were separated by only 1 hour (28), such a mechanism could be involved in other models of conditioning. Finally, although the present study provided no evidence that passive stretch conditioning decreases initial injury following lengthening contractions, the possibility that passive stretch can influence mechanical damage at a time later than what was measured in this study has not been eliminated.

In contrast to adaptations providing protection from the initial mechanical damage that would have to be in place prior to administration of the lengthening contractions, mechanisms activated by passive stretches that protect against secondary injury could impact the injury process at any time between administration of passive stretches and 3 days following the lengthening contractions, when injury was evaluated. Training with passive stretches not only decreases force deficit and morphological damage, but also reduces the number of neutrophils and macrophages that infiltrate following a subsequent bout of lengthening contractions (29). Prior training with passive stretches is sufficient to reduce the number of neutrophils in old mice following lengthening contractions, even though old mice have greater accumulation of neutrophils in comparison to young mice (17). Inhibition of neutrophil infiltration decreases functional and histological indices of damage and hastens recovery following lengthening contractions (30). The mechanism by which training with passive stretches decreases neutrophil accumulation following a subsequent bout of lengthening contractions and if this reduction in neutrophils is associated with the protection provided by passive stretches is unknown. Administration of passive stretches alone causes significant infiltration of neutrophils despite the lack of overt morphological damage (29). Neutrophils release free radicals, proteases, growth factors, cytokines, and chemokines (reviewed in 31,32) which could function as signals for the induction of protective mechanisms. Mechanical loading of cultured myotubes promotes the chemotaxis and priming of neutrophils for release of reactive oxygen species in the absence of injury (33). Further investigation is clearly warranted regarding the signals that lead to neutrophil infiltration following passive stretches and whether such infiltration plays a role in reducing secondary injury.

Conclusion
Passive stretches administered only 1 hour prior to lengthening contractions reduced the force deficit and morphological damage observed in both young and old mice. The protection provided by passive stretch does not appear to be due to decreased initial mechanical damage, and therefore may be caused by a lessened inflammatory response. Because the aging population is highly susceptible to contraction-induced injury, passive stretch conditioning could function as a low-risk form of training sufficient to reduce injury. Passive stretches could also serve as a bridge to other types of more rigorous exercise after an initial baseline of protection is achieved. Better understanding of the mechanism by which passive stretches protect against contraction-induced injury would enhance our ability to design more safe and effective treatments for protecting elderly people from the pain, loss of mobility, and weakness caused by muscle injury.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Financial support was provided by National Institute on Aging grant AG-20591.

We thank Cheryl Hassett and Carol Davis for their help with in vitro force measurements.


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received August 4, 2005

Accepted October 13, 2005


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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G. S. Lynch, J. A. Faulkner, and S. V. Brooks
Force deficits and breakage rates after single lengthening contractions of single fast fibers from unconditioned and conditioned muscles of young and old rats
Am J Physiol Cell Physiol, July 1, 2008; 295(1): C249 - C256.
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