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The deleterious effect of tetanic contraction on rabbit's triceps surae muscle during cyclic loading
Clinical Riomechunics Vol. 11, No. 1, pp. 46-50, 1996 Copyright @ 1995 Elscvier Science Limited Printed in Great Britain. All rights reserved 0268-0033/96 $15.00 + 0.00 ELSEVIER
J-S Sun MD’, Y-H Tsuang MD Pm’, Y-S Hang W W-L Lee MS’, C-K Cheng Pm* ‘Department Biomedical ROC
MD’,
T-K Liu
MD’,
of Orthopedic Surgery, National Taiwan University Hospital, 2Center for Engineering, College of Medicine, National Taiwan University, Taipei, Taiwan,
Summary-To elucidate the effect of nerve stimulation on the mechanical property of muscle-tendon unit, the triceps surae muscle of New Zealand White rabbit was tested with material testing machine. After anaesthesia, the triceps surae muscle-tendon unit of rabbit was tested with cyclic loading during nerve stimulation. The differences between initial and residual force disappeared in the presence of low amplitude and low frequency nerve stimptation. Loading energy applied to the muscle-tendon unit did not change, but the unloading energy increased and energy loss decreased significantly. However, the residual force appeared again at the tetanic stimulation; the loading energy, unloading energy and energy loss all increased significantly. R&?w#rce--intensive exercise training can result in muscle damage and muscle soreness, espesi&tly when the exercise involves eccentric contraction. However, the mechanism responsible forinjury during eccentric contraction is-still unknown. In the present study, we demonstrate that low amplitudes and frequencies of nerve-stimulation have a protective effect on the muscle-tendon unit. However, at the tetanic stimulation, the deleterious effect upon the muscle-tendon unit appeared. This fact may contribute to a better understanding of the mechanism responsible for injury during eccentric contraction. Key words: Nerve stimulation, energy changes, skeletal muscles Clin.
Biomech.
Vol. 11, No. 1, 46-50,
1996
Introduction Muscle is the only tissue in the musculoskeletal system capable of actively developing tension. When skeletal muscle is stimulated, it is rapidly changing from passive tissue into active tissue. Muscle-tendon units contract innumerable times daily. During the activities of daily living, musculoskeletal system is subjected to a wide range of joint motion. Both muscle and tendon components are susceptible to trauma or wear and tear. In sports medicine, stretching exercise has been recommended to prevent injury’.* and to improve performance”. However, intensive exercise training can also result in muscle damage and muscle soreness, especially when the exercise involves eccentric conRecnved: 37 .June 1994: Accepted: 28 June 1995 Correspondence und reprints requests to: Yang-Hwei Tsuang MD
Ph.D, Department of Orthopedic Surgery, National Taiwan University HospiGai. 7, Chung-Shari South Road, Taipei, Taiwan, 100. ROC
tractionlP6. McCully and Faulkner’ demonstrated that the extent of injury was related to the peak force developed during lengthening contraction. However, the mechanism responsible for injury during eccentric contraction is still not well defined. In order to elucidate the effect of the nerve activation on the cyclic loading of skeletal muscle, different amplitudes and frequencies of nerve stimulation were applied when the muscle-tendon unit received cyclic loading. We hypothesized that functioning nerve would affect the mechanical property of muscle-tendon unit during cyclic loading. Methods Twenty New Zealand White rabbits (mean weight: 2.5 kg; SD, 0.2 kg) were divided into two groups. The only anaesthetic used was ketamine, which had no muscle-relaxant effect* and preserved nerve function in the experimental limb. The operation procedure was
Sun et al.: Nerve effect on skeletal
the same as the previous report’. Briefly, an incision on the lateral aspect of each hind limb was made from the mid-calf to the plantar surface of foot. The Achilles tendon was isolated with special care of maintaining the neurovascular supply and the tendon insertion intact. For determining the in situ muscle length, a dial caliper (accurate to 0.05 mm) was used to measure the distance between the origin of the triceps surae at the femur and the insertion at the calcaneus with the knee and the ankle at 90” angulation. Then the anaesthetized rabbit was placed in a frame attached to the MTS machine (MTS BionixTM 858 Test System). The hind limb was immobilized with a K-wire transfixation through the proximal tibia. The distal tendon insertion was freed by osteotomization at the calcaneal tuberosity and clamped to the MTS load cell (MTS 458.20, Microconsole, Axial). A 3-N preload was placed on the muscle, and then the muscle length was again measured’. A skin incision over bilateral buttock region was made to expose the sciatic nerve. Then the sciatic nerve was isolated and clamped with nerve stimulator (TENS SkylarkTM transcutaneous electrical nerve stimulators, Skylark Device Co. Ltd, ROC). In the rabbits of Group I the triceps surae muscle-tendon unit was stretched at a constant rate (5 cm min-‘) to 20% strain of the in situ muscle length. During the passive stretching, various amplitudes of nerve stimulation (pulse width: 120 vs, frequency: 2 Hz, amplitude: 0, 20, 40, 60, 80 mA) was applied in a sequential order. Once the peak strain (20% strain) was reached, the stretching was discontinued and the muscle-tendon unit was returned to its initial resting length. A rest period of 15 min was given between each cycle of loading. In the other hind limb, the muscle-tendon unit was stretched to the same strain (20%) but with various frequencies of nerve stimulation (pulse width: 120 ps, amplitude: 20 mA, frequency: 0, 2, 6, 20, 50 Hz in a sequential order). In the pilot study, maximal load of the muscletendon unit was obtained at the 50 Hz stimulation, we defined this as tetanic nerve stimulation. In Group II the rabbits received the same tests except that the maximal strain was 12%. The changes in force and length required to deform the muscle was simultaneously recorded on a personal computer by the TestlinkTM system Software (PCLABTM Data Translation, Data Translation Inc.). All muscles were kept moist and at physiological temperatures using warm normal saline irrigation. Additional anaesthetic was given as needed. All experimental procedures were performed in accordance with the guidelines set out by the National Institutes of Health policy on the use and care of animals in research. Force and length were plotted to generate a ‘hysteresis’ loop (Figure 1). The initial force depicted the preload of the testing muscle-tendon unit before each testing cycle. The maximal load attained during each testing cycle was indicated as peak force. The load at the end of each testing cycle when the muscletendon unit returned to its initial length was designed as the residual force. The portion of area under the loop
Load
muscles
47
B
Deformation Figure 1. Load-deformation curve for cyclic testing of material. A: initial force, B: peakfdrce, C: residualforce. Loading energy: area below the ..._ curve A-B; unloading energy: area below curve B-C; energy loss. loading energy- unloading energy.
before the maximal force attained (A to B in Figure 1) was calculated and indicated as the loading energy applied to the muscle-tendon unit. The area after the maximal force to initial resting length (B to C in Figure 1) indicated the unloading energy. The included area indicates the energy loss associated with the viscous retardation. The loading energy, unloading energy and energy loss were evaluated by a repeated measurement of ANOVA statistic method. The post hoc tests performed was Bonferroni’s t test. The initial force before the ‘hysteresis’ loop and final residual force at the end of the loop after various tests were recorded. The difference between these two forces was compared by paired t test. The level of statistical significance was set at 5%. Results Effect of stimulation amplitude
The difference between the initial and residual force at 20%-strain test disappeared after different amplitudes nerve stimulation. The unloading energy of muscletendon unit increased and energy loss decreased (Tables 1, 2). Force changes
The difference between the initial forces and the residual forces were statistically significant in the group of 20% strain without nerve stimulation (P
Clin. Biomech. Vol. 11, No. 1, 1996
46
Table 1. Effect of stimulation in := 10) Amplit. ImA)
of Stim.
Gmup I 0
Initial NV)
amplitude
F.
on the initial and residual
Residual /Nl
F.
forces.
P
4).
20 40 60 80 P
(at 20% strain) 3.66 (SD, 0.38) 3.60 (SD, 0.26) 3.43 (SD, 0.26) 3.54 (SD, 0.40) 3.40 (SD, 0.21) NS
3.22 3.45 3.32 3.63 3.58 NS
(SD, 0.44) (SD, 0.41) (SD, 0.17) (SD, 0.30) (SD, 0.61)
0.005 NS NS NS NS
Group Ii 0 2 4 6 8 P
iat 12% 3.51 (SD, 3.52 (SD, 3.75 (SD, 3.60 (SD, 3.60 (SD, NS
3.46 3.38 3.46 3.46 3.70 NS
(SD,
0.53) (SD, 0.36) (SD, 0.43) (SD, 0.34)
NS NS NS NS NS
strain) 0.30) 0.35 0.74) 0.3)
0.30)
(SD, 0.49)
residual force, peak force, loading energy, unloading energy, and energy loss were all significantly affected by higher frequencies of nerve stimulation (Tables 3,
Force changes
In the trial of 20% strain without nerve stimulation, the difference between the initial force and the residual force was also statistically significant (P~O.005). At either 12% or 20% strain, the differences between the initial forces and the residual forces were statistically significant in the group with 50 Hz nerve stimulation only (P
NS, not signifkzant. data were analysed by paired t test
AH
12% strain, there was significant difference existed in peak force (Table 2). The difference existed between the non-stimulated group (0 mA) and the stimulated groups (20, 40, 60, 80 mA), but not among the stimulated groups. Energy changes
When stretching to 20% strain, the difference of loading energy as well as the difference of unloading energy applied to the muscle-tendon unit were not statistically significant. However, the difference in energy loss was statistically significant (Table 2). The difference (PcO.01) existed between the nonstimulated group (0 mA) and the stimulated groups (20, 40, 60,- 80 mA), but nqt among the stimulated groups. Similar effects were observed at the 12% strain except that unloading energy also increased significantly (P
Energy changes
The frequency of nerve stimulation can affect the muscle-tendon unit significantly. The initial force,
At 20% strain, the differences of loading energy, unloading energy, and energy loss among various frequencies of nerve stimulation were statistically significant (P
T&e
of muscle-tendon
of stimutution
effect
2. Effect of stimulation
Amplit. lmAj
of Stim.
Group 0 20 40 60 80 P
1
Group 0 20 40 60 80 P
II
frequency
amplitude
to the peak force and energy
changes
Peak force O’J)
Loading /N mm)
(at 20% strain) 113.96 (SD, 21.63) 122.79 (SD, 27.09) 127.71 (SD, 25.99) 127.78 (SD, 32.07) 128.09 (SD, 29.31) NS
598.50 604.34 583.98 598.54 620.30 NS
159.28) 154.26) 146.26) (so, 162.06) (so, 167.58)
(at 12% strain) 28.79 (SD, 7.27) 43.30 (SD, 13.86) 46.39 (SD, 16.17) 47.81 (SD, 18.04) 49.36 (SD, 17.23) 0.03’
144.02 141.12 142.66 146.32 149.98 NS
15.00) 16.88) 19.84) (SD, 23.16) (SD, 20.36)
energy
(SD, (SD, (SD,
unit. (n = 10) Unloading IN mm) 382.60 487.56 475.66 486.14
65.98) 95.88) (SD, 91.58) (SD, 104.36)
493.54
(SD, 115.90)
(SD, (SD,
NS (SD, (SD, (SD,
107.50 125.54 129.84 133.82 136.48 0.0007*
data were anetysed by repeated measurement of ANOVAtest. NS, not significant. *The difference (P~0.0)) existed between the non-stimulated group LOmA) end the stimulated groups (20, 40, 60. 80 mA).
All
energy
(SD, 9.50) (SD, 12.08) (SD, 16.38) (SD, 18.56) (SD, 16.72)
Energy loss (N mm) 215.90 116.86 108.32 112.42 122.30 0.02’
(SD, 107.60) (SD, 68.46) (SD, 63.48) (SD, 65.70) (SD, 64.36)
36.52 (SD, 8.30) 15.56 (SD, 6.46) 13.26 (SD, 9.10) 12.50 (SD, 7.34) 13.60 (SD, 5.02) PC 0.0001*
Sun et al.: Nerve effect on skeletal Table 3. Effect of stimulation (n = 10)
frequency
Freq. of Stim. IHA
Initial fh’)
Group I 0 2 6 20 50 P
(at 20% strain) 3.64 (SD, 0.54) 3.51 (SD, 0.44) 3.51 (SD, 0.34) 3.61 (SD, 0.32) 8.04 (SD, 2.17) <0.0001
Group II 0 2 6 20 50 P
(at 12% strain) 3.49 LSD, 0.29) 3.42 (SD, 0.21 3.53 (SD, 0.20) 4.40 (SD, 1.01) 8.34 (SD, 2.58) <0.0001
NS, not significant. All data were analyzed
F.
by paired
on the initial and residual
Residual IN) 3.21 3.40 3.52 3.53 4.20
F.
0.43) 0.40) (SD, 0.36) (SD, 0.28) (SD, 1.01) (SD, (SD,
forces.
P
0.004 NS NS NS 0.02
0.001
3.38 (SD, 3.41 (SD, 3.46 (SD, 4.50 (SD, 4.77 (SD, 0.004
0.33) 0.32) 0.24) 0.72) 1.13)
NS NS NS NS 0.0003
r test
(2 Hz and 6 Hz) were not significant. Similar effects were observed at the 12% strain except the differences of unloading energy were not statistically significant between the trials of 20 Hz and 50 Hz of nerve stimulation (BO.05). However, there were statistically significant differences existing among 20 Hz and 50 Hz with other trials (P
60
20 0 0
5
10
15
20
Strain (%I Figure 2. The load-deformation curves produced by applying five different frequencies of nerve stimulation (0 Hz, 2 Hz, 6 Hz, 20 Hz, 50 Hz) cycle to a triceps surae muscle-tendon unit. For clarity, only the loops with 2,50 Hz nerve stimulation were depicted. Note that there was similarity existing between the hysteretic loops of non-stimulated (0 Hz) and low-frequency stimulated curves (2 Hz). At tetanic stimulation (50 Hz), the loading curve changed from a concave shape to a convex shape and the loop became elliptical.
muscles
49
fact that the loss of nerve function significantly reduced the peak force and the energy absorption before peak force”. However, the above-mentioned studies based on the tests that specimens were loaded to rupture by a single loading test. No unloading phase was performed before rupture. In most activity of the daily living, the repetitive contraction-relaxation cycles of muscletendon unit were much similar to a dynamic cyclic loading. We designed this study to test the nerve effect on cyclic loading of the muscle-tendon unit. In this study, there was a statistically significant difference between initial force and residual force in the group of 20% strain without nerve stimulation. When nerve stimulation was applied, the differences disappeared with the exception of tetanic stimulation (Tables 1, 3). Simi 1ar result was observed in the group of 12% strain. The nerve has a role in the regulation of reversible plasticity of the skeletal muscle. When the nerve stimulation was applied, the difference in the initial and residual force disappeared with the exception of tetanic stimulation (Table 3). The amplitude of nerve stimulation did not affect the peak force attained at higher strain. However, at lower strain (12% in this study), there was a significant difference existing in peak force (Table 2). The peak force of the muscle-tendon unit without nerve stimulation was relatively low in the 12% strain. Potentiation of the muscle by nerve stimulation can affect the magnitude of peak force significantly (Table 3). At 12% or 20% strain, the peak force attained at 20 Hz and 50 Hz nerve stimulation was significantly higher than the non-stimulated or lower frequencies’ nerve stimulated muscle-tendon unit. If tetanic nerve stimulation was applied, the peak force of muscle-tendon unit increased significantly, the possibility to injure the muscle-tendon unit increased (Table 4). The loading energy might work as to deformed or even to destroy the muscle-tendon unit (if it exceeds the yield point), the unloading energy is the energy exerted upon the surrounding medium during recovering the muscle-tendon unit to its initial resting condition, and the energy loss represents the energy that dissipated out of muscle-tendon unit during cyclic loading. In this study, the unloading energy but not loading energy was increased and the energy loss decreased at low amplitude nerve stimulation (Table 3). Low frequency nerve stimulation (2 Hz in this study) might help the muscle-tendon unit recovering to its original resting condition. The beneficial effects of nerve-stimulation were even more effective at the lower strain within the viscoelastic component of the muscle-tendon unit (at 12% strain in this study). It suggests that existence of nerve may help to protect the muscle-tendon unit from injury. During tetanic stimulation, there was significant increase in energy loss with a change of the curve pattern (Figure 2). Although the unloading energy was also increased significantly, the increase in the energy loss was mainly due to the larger increase in amount of the loading energy (Table 4). When tetanic nerve stimulation was applied, the loading energy and energy
50
Clin. Biomech.
Table 4. Effect of stimulation
Vol. 11, No. 1, 1996 frequency
to the peak force and energy
changes
f%?cj. of sth?. (Hz) ----.__l_
Peak force /N/
Loading lN mml
Group 0
(at 20% strain) 117.90 (SD, 17.09) 124.29 (SD, 16.09) 130.24 (SD, 21.72) 139.39 (SD, 23.55)
624.30 589.44 614.70 959.48
1
2 6
20 50
152.09
P
Group 0 2 6 20 5G P All
II
(SD, 30.92)
1234.74
of muscle-tendon
unit. (n = 10) Unloading NV mm)
energy
(SD, 167.28)
414.10 457.58 483.56 618.46 572.70 0.008'"
(SD, 137.74) (SD, 165.78) (SD, 245. IO) (SD, 275.94)
0.02*
P<0.0001*
(at 12% strain) 28.58 (SD, 4.19) 38.90 (SD, 11.22) 39.34 (SD, 14.88) 57.33 (SD, 25.92) 59.15 (SO, 19.26) 0.0005****
149.84 (SD, 144.40 (SD, 142.72 (SD, 352.02 (SD, 487.08 (SD, P<0.0001*
energy
Energy loss IN mml
(SD, 102.76) 107.94) (SD, 116.08) (SD, 159.06) (SD, 133.84)
210.20 (SD, 69.36) 131.84 (SD, 36.68) 131.14 (SD, 65.88) 341.02 (SD, 139.36) 662.04 (SD, 198.28) P<0.0001***
(SD,
36.34 (SD, 7.00) 16.96 (SD, 6.82) 16.10 (SD, 7.84) 112.40 (SD, 50.72) 249.00 (so, 85.46) P<0.0001***
113.50 (SD, 10.96) 125.64 (SD, 11.04) 130.02 (SD, 18.54) 243.62 (SD, 62.14) 238.08 (SD, 65.54) P<0.0001****
10.96) 16.38) 19.54) 108.72) 146.30)
data were analysed by repeated measurement of ANOVArest.
*lMference existed beWeen 20 Hz and 50 Hz stimulation ‘*Difference was not statistically significant only between iP
and among 20 Hz and 50 Hz with other variables iPcO.01). 20 and 50 Hz of nerve stimulation. There were statistically
significant
differences
existing
between
6 Hz stimulation. The differences between other variables were statistically significant (P~0.05). the trials of 20 Hz and 50 Hz of nerve stimulation (P20.05). Otherwise, there were statistically 0.05).
loss increased significantly and the possibility to injure the muscle-tendon unit increased. It was not surprising that the residual force (or residual deformation) changed significantly after tetanic nerve stimulation (Table 3). Since stretching of muscle was an effective stimulus for contraction, there is some gross simulation between the curves without and with nerve stimulation. During tetanic contraction, there is significant increase in energy loss within the hysteresis loop and changes in curve pattern. The effect of low frequency nerve stimulation didn’t increase the loading energy but increase the unloading energy. It suggests that low frequency nerve stimulation may help muscle-tendon unit recovering to its original resting condition. However, high frequency nerve stimulation, especially tetanic stimulation (SO Hz in this study), loading energy increased more than the unloading energy did. As a result, energy loss increased, and the deleterious effects of high frequency nerve stimulation appeared. This study showed that nerve function in viva can protect muscles from injury by decreasing the energy loss. Acknowkdganents The authors sincerely thank the National Science Council (ROC) for their financial support of this research. We dedicate this paper with gratitude to the National Science Council, ROC.
other
significant
frequencies
differences
References 1 Ciullo JV, Zarins B. Biomechanics of the
musculotendinous unit: relation to athletic performance and injury. Clin Sports Med 1983; 2: 71-86 2 Ekstrand J, Gillquist J. The avoidability of soccer injuries. Int J Sports Med 1983; 4: 124-8 3 Stanish WD. Neurophysiology of stretching. In: D’Ambrosia R, Drez D, edB. Prevention and Treatment of Running Injuries. Slack, NJ, 1982; 135-45 4 Schwane JA, Johnson SR, Vandenakker CB, Armstrong
5 6 7 8 9 10
RB. Delayed-onset muscular soreness and plasma CPK and LDH activities after downhill running. Med Sci Sports Exert 1983; 15: 51-6 Armstrong RB. Mechanism of exercise-induced delayed onset muscular soreness: a brief review. Med Sci Sports Exert 1984; 16: 529-38 Evans WJ, Meredith CN, Cannon JG et al. Metabolic changes following eccentric exercise in trained and untrained men. J Appl Physioll985; 61: 1863- 8 MuCully KK, Faulkner JA. Characteristics of lengthening contractions associated with injury to skeletal muscle fibers. JAppl Physioll986; 61: 293-9 Wingard LBJr, Brody TM, Larner J, et al. Human Pharmacology: Molecular to Clinical. Wolfe, ISE. London, 1991; 413-32 Sun JS, Tsuang YH, Liu TK et al. Failure sites and peak tensile forces of the composite triceps surae muscle by passive extension in rabbit. Clin Biomech 1994; 9: 310- 14 Sun JS, Tsuang YH, Cheng CK et al. The effect of nerve function on the failure mechanism of the triceps surae muscle by passive extension in the rabbit. J Formosan MedAssoc 1994;93:51-5
J-S Sun MD’, Y-H Tsuang MD Pm’, Y-S Hang W W-L Lee MS’, C-K Cheng Pm* ‘Department Biomedical ROC
MD’,
T-K Liu
MD’,
of Orthopedic Surgery, National Taiwan University Hospital, 2Center for Engineering, College of Medicine, National Taiwan University, Taipei, Taiwan,
Summary-To elucidate the effect of nerve stimulation on the mechanical property of muscle-tendon unit, the triceps surae muscle of New Zealand White rabbit was tested with material testing machine. After anaesthesia, the triceps surae muscle-tendon unit of rabbit was tested with cyclic loading during nerve stimulation. The differences between initial and residual force disappeared in the presence of low amplitude and low frequency nerve stimptation. Loading energy applied to the muscle-tendon unit did not change, but the unloading energy increased and energy loss decreased significantly. However, the residual force appeared again at the tetanic stimulation; the loading energy, unloading energy and energy loss all increased significantly. R&?w#rce--intensive exercise training can result in muscle damage and muscle soreness, espesi&tly when the exercise involves eccentric contraction. However, the mechanism responsible forinjury during eccentric contraction is-still unknown. In the present study, we demonstrate that low amplitudes and frequencies of nerve-stimulation have a protective effect on the muscle-tendon unit. However, at the tetanic stimulation, the deleterious effect upon the muscle-tendon unit appeared. This fact may contribute to a better understanding of the mechanism responsible for injury during eccentric contraction. Key words: Nerve stimulation, energy changes, skeletal muscles Clin.
Biomech.
Vol. 11, No. 1, 46-50,
1996
Introduction Muscle is the only tissue in the musculoskeletal system capable of actively developing tension. When skeletal muscle is stimulated, it is rapidly changing from passive tissue into active tissue. Muscle-tendon units contract innumerable times daily. During the activities of daily living, musculoskeletal system is subjected to a wide range of joint motion. Both muscle and tendon components are susceptible to trauma or wear and tear. In sports medicine, stretching exercise has been recommended to prevent injury’.* and to improve performance”. However, intensive exercise training can also result in muscle damage and muscle soreness, especially when the exercise involves eccentric conRecnved: 37 .June 1994: Accepted: 28 June 1995 Correspondence und reprints requests to: Yang-Hwei Tsuang MD
Ph.D, Department of Orthopedic Surgery, National Taiwan University HospiGai. 7, Chung-Shari South Road, Taipei, Taiwan, 100. ROC
tractionlP6. McCully and Faulkner’ demonstrated that the extent of injury was related to the peak force developed during lengthening contraction. However, the mechanism responsible for injury during eccentric contraction is still not well defined. In order to elucidate the effect of the nerve activation on the cyclic loading of skeletal muscle, different amplitudes and frequencies of nerve stimulation were applied when the muscle-tendon unit received cyclic loading. We hypothesized that functioning nerve would affect the mechanical property of muscle-tendon unit during cyclic loading. Methods Twenty New Zealand White rabbits (mean weight: 2.5 kg; SD, 0.2 kg) were divided into two groups. The only anaesthetic used was ketamine, which had no muscle-relaxant effect* and preserved nerve function in the experimental limb. The operation procedure was
Sun et al.: Nerve effect on skeletal
the same as the previous report’. Briefly, an incision on the lateral aspect of each hind limb was made from the mid-calf to the plantar surface of foot. The Achilles tendon was isolated with special care of maintaining the neurovascular supply and the tendon insertion intact. For determining the in situ muscle length, a dial caliper (accurate to 0.05 mm) was used to measure the distance between the origin of the triceps surae at the femur and the insertion at the calcaneus with the knee and the ankle at 90” angulation. Then the anaesthetized rabbit was placed in a frame attached to the MTS machine (MTS BionixTM 858 Test System). The hind limb was immobilized with a K-wire transfixation through the proximal tibia. The distal tendon insertion was freed by osteotomization at the calcaneal tuberosity and clamped to the MTS load cell (MTS 458.20, Microconsole, Axial). A 3-N preload was placed on the muscle, and then the muscle length was again measured’. A skin incision over bilateral buttock region was made to expose the sciatic nerve. Then the sciatic nerve was isolated and clamped with nerve stimulator (TENS SkylarkTM transcutaneous electrical nerve stimulators, Skylark Device Co. Ltd, ROC). In the rabbits of Group I the triceps surae muscle-tendon unit was stretched at a constant rate (5 cm min-‘) to 20% strain of the in situ muscle length. During the passive stretching, various amplitudes of nerve stimulation (pulse width: 120 vs, frequency: 2 Hz, amplitude: 0, 20, 40, 60, 80 mA) was applied in a sequential order. Once the peak strain (20% strain) was reached, the stretching was discontinued and the muscle-tendon unit was returned to its initial resting length. A rest period of 15 min was given between each cycle of loading. In the other hind limb, the muscle-tendon unit was stretched to the same strain (20%) but with various frequencies of nerve stimulation (pulse width: 120 ps, amplitude: 20 mA, frequency: 0, 2, 6, 20, 50 Hz in a sequential order). In the pilot study, maximal load of the muscletendon unit was obtained at the 50 Hz stimulation, we defined this as tetanic nerve stimulation. In Group II the rabbits received the same tests except that the maximal strain was 12%. The changes in force and length required to deform the muscle was simultaneously recorded on a personal computer by the TestlinkTM system Software (PCLABTM Data Translation, Data Translation Inc.). All muscles were kept moist and at physiological temperatures using warm normal saline irrigation. Additional anaesthetic was given as needed. All experimental procedures were performed in accordance with the guidelines set out by the National Institutes of Health policy on the use and care of animals in research. Force and length were plotted to generate a ‘hysteresis’ loop (Figure 1). The initial force depicted the preload of the testing muscle-tendon unit before each testing cycle. The maximal load attained during each testing cycle was indicated as peak force. The load at the end of each testing cycle when the muscletendon unit returned to its initial length was designed as the residual force. The portion of area under the loop
Load
muscles
47
B
Deformation Figure 1. Load-deformation curve for cyclic testing of material. A: initial force, B: peakfdrce, C: residualforce. Loading energy: area below the ..._ curve A-B; unloading energy: area below curve B-C; energy loss. loading energy- unloading energy.
before the maximal force attained (A to B in Figure 1) was calculated and indicated as the loading energy applied to the muscle-tendon unit. The area after the maximal force to initial resting length (B to C in Figure 1) indicated the unloading energy. The included area indicates the energy loss associated with the viscous retardation. The loading energy, unloading energy and energy loss were evaluated by a repeated measurement of ANOVA statistic method. The post hoc tests performed was Bonferroni’s t test. The initial force before the ‘hysteresis’ loop and final residual force at the end of the loop after various tests were recorded. The difference between these two forces was compared by paired t test. The level of statistical significance was set at 5%. Results Effect of stimulation amplitude
The difference between the initial and residual force at 20%-strain test disappeared after different amplitudes nerve stimulation. The unloading energy of muscletendon unit increased and energy loss decreased (Tables 1, 2). Force changes
The difference between the initial forces and the residual forces were statistically significant in the group of 20% strain without nerve stimulation (P
Clin. Biomech. Vol. 11, No. 1, 1996
46
Table 1. Effect of stimulation in := 10) Amplit. ImA)
of Stim.
Gmup I 0
Initial NV)
amplitude
F.
on the initial and residual
Residual /Nl
F.
forces.
P
4).
20 40 60 80 P
(at 20% strain) 3.66 (SD, 0.38) 3.60 (SD, 0.26) 3.43 (SD, 0.26) 3.54 (SD, 0.40) 3.40 (SD, 0.21) NS
3.22 3.45 3.32 3.63 3.58 NS
(SD, 0.44) (SD, 0.41) (SD, 0.17) (SD, 0.30) (SD, 0.61)
0.005 NS NS NS NS
Group Ii 0 2 4 6 8 P
iat 12% 3.51 (SD, 3.52 (SD, 3.75 (SD, 3.60 (SD, 3.60 (SD, NS
3.46 3.38 3.46 3.46 3.70 NS
(SD,
0.53) (SD, 0.36) (SD, 0.43) (SD, 0.34)
NS NS NS NS NS
strain) 0.30) 0.35 0.74) 0.3)
0.30)
(SD, 0.49)
residual force, peak force, loading energy, unloading energy, and energy loss were all significantly affected by higher frequencies of nerve stimulation (Tables 3,
Force changes
In the trial of 20% strain without nerve stimulation, the difference between the initial force and the residual force was also statistically significant (P~O.005). At either 12% or 20% strain, the differences between the initial forces and the residual forces were statistically significant in the group with 50 Hz nerve stimulation only (P
NS, not signifkzant. data were analysed by paired t test
AH
12% strain, there was significant difference existed in peak force (Table 2). The difference existed between the non-stimulated group (0 mA) and the stimulated groups (20, 40, 60, 80 mA), but not among the stimulated groups. Energy changes
When stretching to 20% strain, the difference of loading energy as well as the difference of unloading energy applied to the muscle-tendon unit were not statistically significant. However, the difference in energy loss was statistically significant (Table 2). The difference (PcO.01) existed between the nonstimulated group (0 mA) and the stimulated groups (20, 40, 60,- 80 mA), but nqt among the stimulated groups. Similar effects were observed at the 12% strain except that unloading energy also increased significantly (P
Energy changes
The frequency of nerve stimulation can affect the muscle-tendon unit significantly. The initial force,
At 20% strain, the differences of loading energy, unloading energy, and energy loss among various frequencies of nerve stimulation were statistically significant (P
T&e
of muscle-tendon
of stimutution
effect
2. Effect of stimulation
Amplit. lmAj
of Stim.
Group 0 20 40 60 80 P
1
Group 0 20 40 60 80 P
II
frequency
amplitude
to the peak force and energy
changes
Peak force O’J)
Loading /N mm)
(at 20% strain) 113.96 (SD, 21.63) 122.79 (SD, 27.09) 127.71 (SD, 25.99) 127.78 (SD, 32.07) 128.09 (SD, 29.31) NS
598.50 604.34 583.98 598.54 620.30 NS
159.28) 154.26) 146.26) (so, 162.06) (so, 167.58)
(at 12% strain) 28.79 (SD, 7.27) 43.30 (SD, 13.86) 46.39 (SD, 16.17) 47.81 (SD, 18.04) 49.36 (SD, 17.23) 0.03’
144.02 141.12 142.66 146.32 149.98 NS
15.00) 16.88) 19.84) (SD, 23.16) (SD, 20.36)
energy
(SD, (SD, (SD,
unit. (n = 10) Unloading IN mm) 382.60 487.56 475.66 486.14
65.98) 95.88) (SD, 91.58) (SD, 104.36)
493.54
(SD, 115.90)
(SD, (SD,
NS (SD, (SD, (SD,
107.50 125.54 129.84 133.82 136.48 0.0007*
data were anetysed by repeated measurement of ANOVAtest. NS, not significant. *The difference (P~0.0)) existed between the non-stimulated group LOmA) end the stimulated groups (20, 40, 60. 80 mA).
All
energy
(SD, 9.50) (SD, 12.08) (SD, 16.38) (SD, 18.56) (SD, 16.72)
Energy loss (N mm) 215.90 116.86 108.32 112.42 122.30 0.02’
(SD, 107.60) (SD, 68.46) (SD, 63.48) (SD, 65.70) (SD, 64.36)
36.52 (SD, 8.30) 15.56 (SD, 6.46) 13.26 (SD, 9.10) 12.50 (SD, 7.34) 13.60 (SD, 5.02) PC 0.0001*
Sun et al.: Nerve effect on skeletal Table 3. Effect of stimulation (n = 10)
frequency
Freq. of Stim. IHA
Initial fh’)
Group I 0 2 6 20 50 P
(at 20% strain) 3.64 (SD, 0.54) 3.51 (SD, 0.44) 3.51 (SD, 0.34) 3.61 (SD, 0.32) 8.04 (SD, 2.17) <0.0001
Group II 0 2 6 20 50 P
(at 12% strain) 3.49 LSD, 0.29) 3.42 (SD, 0.21 3.53 (SD, 0.20) 4.40 (SD, 1.01) 8.34 (SD, 2.58) <0.0001
NS, not significant. All data were analyzed
F.
by paired
on the initial and residual
Residual IN) 3.21 3.40 3.52 3.53 4.20
F.
0.43) 0.40) (SD, 0.36) (SD, 0.28) (SD, 1.01) (SD, (SD,
forces.
P
0.004 NS NS NS 0.02
0.001
3.38 (SD, 3.41 (SD, 3.46 (SD, 4.50 (SD, 4.77 (SD, 0.004
0.33) 0.32) 0.24) 0.72) 1.13)
NS NS NS NS 0.0003
r test
(2 Hz and 6 Hz) were not significant. Similar effects were observed at the 12% strain except the differences of unloading energy were not statistically significant between the trials of 20 Hz and 50 Hz of nerve stimulation (BO.05). However, there were statistically significant differences existing among 20 Hz and 50 Hz with other trials (P
60
20 0 0
5
10
15
20
Strain (%I Figure 2. The load-deformation curves produced by applying five different frequencies of nerve stimulation (0 Hz, 2 Hz, 6 Hz, 20 Hz, 50 Hz) cycle to a triceps surae muscle-tendon unit. For clarity, only the loops with 2,50 Hz nerve stimulation were depicted. Note that there was similarity existing between the hysteretic loops of non-stimulated (0 Hz) and low-frequency stimulated curves (2 Hz). At tetanic stimulation (50 Hz), the loading curve changed from a concave shape to a convex shape and the loop became elliptical.
muscles
49
fact that the loss of nerve function significantly reduced the peak force and the energy absorption before peak force”. However, the above-mentioned studies based on the tests that specimens were loaded to rupture by a single loading test. No unloading phase was performed before rupture. In most activity of the daily living, the repetitive contraction-relaxation cycles of muscletendon unit were much similar to a dynamic cyclic loading. We designed this study to test the nerve effect on cyclic loading of the muscle-tendon unit. In this study, there was a statistically significant difference between initial force and residual force in the group of 20% strain without nerve stimulation. When nerve stimulation was applied, the differences disappeared with the exception of tetanic stimulation (Tables 1, 3). Simi 1ar result was observed in the group of 12% strain. The nerve has a role in the regulation of reversible plasticity of the skeletal muscle. When the nerve stimulation was applied, the difference in the initial and residual force disappeared with the exception of tetanic stimulation (Table 3). The amplitude of nerve stimulation did not affect the peak force attained at higher strain. However, at lower strain (12% in this study), there was a significant difference existing in peak force (Table 2). The peak force of the muscle-tendon unit without nerve stimulation was relatively low in the 12% strain. Potentiation of the muscle by nerve stimulation can affect the magnitude of peak force significantly (Table 3). At 12% or 20% strain, the peak force attained at 20 Hz and 50 Hz nerve stimulation was significantly higher than the non-stimulated or lower frequencies’ nerve stimulated muscle-tendon unit. If tetanic nerve stimulation was applied, the peak force of muscle-tendon unit increased significantly, the possibility to injure the muscle-tendon unit increased (Table 4). The loading energy might work as to deformed or even to destroy the muscle-tendon unit (if it exceeds the yield point), the unloading energy is the energy exerted upon the surrounding medium during recovering the muscle-tendon unit to its initial resting condition, and the energy loss represents the energy that dissipated out of muscle-tendon unit during cyclic loading. In this study, the unloading energy but not loading energy was increased and the energy loss decreased at low amplitude nerve stimulation (Table 3). Low frequency nerve stimulation (2 Hz in this study) might help the muscle-tendon unit recovering to its original resting condition. The beneficial effects of nerve-stimulation were even more effective at the lower strain within the viscoelastic component of the muscle-tendon unit (at 12% strain in this study). It suggests that existence of nerve may help to protect the muscle-tendon unit from injury. During tetanic stimulation, there was significant increase in energy loss with a change of the curve pattern (Figure 2). Although the unloading energy was also increased significantly, the increase in the energy loss was mainly due to the larger increase in amount of the loading energy (Table 4). When tetanic nerve stimulation was applied, the loading energy and energy
50
Clin. Biomech.
Table 4. Effect of stimulation
Vol. 11, No. 1, 1996 frequency
to the peak force and energy
changes
f%?cj. of sth?. (Hz) ----.__l_
Peak force /N/
Loading lN mml
Group 0
(at 20% strain) 117.90 (SD, 17.09) 124.29 (SD, 16.09) 130.24 (SD, 21.72) 139.39 (SD, 23.55)
624.30 589.44 614.70 959.48
1
2 6
20 50
152.09
P
Group 0 2 6 20 5G P All
II
(SD, 30.92)
1234.74
of muscle-tendon
unit. (n = 10) Unloading NV mm)
energy
(SD, 167.28)
414.10 457.58 483.56 618.46 572.70 0.008'"
(SD, 137.74) (SD, 165.78) (SD, 245. IO) (SD, 275.94)
0.02*
P<0.0001*
(at 12% strain) 28.58 (SD, 4.19) 38.90 (SD, 11.22) 39.34 (SD, 14.88) 57.33 (SD, 25.92) 59.15 (SO, 19.26) 0.0005****
149.84 (SD, 144.40 (SD, 142.72 (SD, 352.02 (SD, 487.08 (SD, P<0.0001*
energy
Energy loss IN mml
(SD, 102.76) 107.94) (SD, 116.08) (SD, 159.06) (SD, 133.84)
210.20 (SD, 69.36) 131.84 (SD, 36.68) 131.14 (SD, 65.88) 341.02 (SD, 139.36) 662.04 (SD, 198.28) P<0.0001***
(SD,
36.34 (SD, 7.00) 16.96 (SD, 6.82) 16.10 (SD, 7.84) 112.40 (SD, 50.72) 249.00 (so, 85.46) P<0.0001***
113.50 (SD, 10.96) 125.64 (SD, 11.04) 130.02 (SD, 18.54) 243.62 (SD, 62.14) 238.08 (SD, 65.54) P<0.0001****
10.96) 16.38) 19.54) 108.72) 146.30)
data were analysed by repeated measurement of ANOVArest.
*lMference existed beWeen 20 Hz and 50 Hz stimulation ‘*Difference was not statistically significant only between iP
and among 20 Hz and 50 Hz with other variables iPcO.01). 20 and 50 Hz of nerve stimulation. There were statistically
significant
differences
existing
between
6 Hz stimulation. The differences between other variables were statistically significant (P~0.05). the trials of 20 Hz and 50 Hz of nerve stimulation (P20.05). Otherwise, there were statistically 0.05).
loss increased significantly and the possibility to injure the muscle-tendon unit increased. It was not surprising that the residual force (or residual deformation) changed significantly after tetanic nerve stimulation (Table 3). Since stretching of muscle was an effective stimulus for contraction, there is some gross simulation between the curves without and with nerve stimulation. During tetanic contraction, there is significant increase in energy loss within the hysteresis loop and changes in curve pattern. The effect of low frequency nerve stimulation didn’t increase the loading energy but increase the unloading energy. It suggests that low frequency nerve stimulation may help muscle-tendon unit recovering to its original resting condition. However, high frequency nerve stimulation, especially tetanic stimulation (SO Hz in this study), loading energy increased more than the unloading energy did. As a result, energy loss increased, and the deleterious effects of high frequency nerve stimulation appeared. This study showed that nerve function in viva can protect muscles from injury by decreasing the energy loss. Acknowkdganents The authors sincerely thank the National Science Council (ROC) for their financial support of this research. We dedicate this paper with gratitude to the National Science Council, ROC.
other
significant
frequencies
differences
References 1 Ciullo JV, Zarins B. Biomechanics of the
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RB. Delayed-onset muscular soreness and plasma CPK and LDH activities after downhill running. Med Sci Sports Exert 1983; 15: 51-6 Armstrong RB. Mechanism of exercise-induced delayed onset muscular soreness: a brief review. Med Sci Sports Exert 1984; 16: 529-38 Evans WJ, Meredith CN, Cannon JG et al. Metabolic changes following eccentric exercise in trained and untrained men. J Appl Physioll985; 61: 1863- 8 MuCully KK, Faulkner JA. Characteristics of lengthening contractions associated with injury to skeletal muscle fibers. JAppl Physioll986; 61: 293-9 Wingard LBJr, Brody TM, Larner J, et al. Human Pharmacology: Molecular to Clinical. Wolfe, ISE. London, 1991; 413-32 Sun JS, Tsuang YH, Liu TK et al. Failure sites and peak tensile forces of the composite triceps surae muscle by passive extension in rabbit. Clin Biomech 1994; 9: 310- 14 Sun JS, Tsuang YH, Cheng CK et al. The effect of nerve function on the failure mechanism of the triceps surae muscle by passive extension in the rabbit. J Formosan MedAssoc 1994;93:51-5