EXPERIMENTAL Sensory Nerve Cross-Anastomosis and Electrical Muscle Stimulation Synergistically Enhance Functional Recovery of Chronically Denervated Muscle Michael P. Willand, Ph.D. Michael Holmes, B.Sc. James R. Bain, M.D. Hubert de Bruin, Ph.D. Margaret Fahnestock, Ph.D. Hamilton, Ontario, Canada
Background: Long-term muscle denervation leads to severe and irreversible atrophy coupled with loss of force and motor function. These factors contribute to poor functional recovery following delayed reinnervation. The authors’ previous work demonstrated that temporarily suturing a sensory nerve to the distal motor stump (called sensory protection) significantly reduces muscle atrophy and improves function following reinnervation. The authors have also shown that 1 month of electrical stimulation of denervated muscle significantly improves function and reduces atrophy. In this study, the authors tested whether a combination of sensory protection and electrical stimulation would enhance functional recovery more than either treatment alone. Methods: Rat gastrocnemius muscles were denervated by cutting the tibial nerve. The peroneal nerve was then sutured to the distal tibial stump following 3 months of treatment (i.e., electrical stimulation, sensory protection, or both). Three months after peroneal repair, functional and histologic measurements were taken. Results: All treatment groups had significantly higher muscle weight (p < 0.05) and twitch force (p < 0.001) compared with the untreated group (denervated), but fiber type composition did not differ between groups. Importantly, muscle weight and force were significantly greater in the combined treatment group (p < 0.05) compared with stimulation or sensory protection alone. The combined treatment also produced motor unit counts significantly greater than sensory protection alone (p < 0.05). Conclusions: The combination treatment synergistically reduces atrophy and improves reinnervation and functional measures following delayed nerve repair, suggesting that these approaches work through different mechanisms. The authors’ research supports the clinical use of both modalities together following peripheral nerve injury. (Plast. Reconstr. Surg. 134: 736e, 2014.)
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eripheral nerve injuries ranging from crush to transection can seriously affect distal muscle structure and function. Longterm denervation is accompanied by irreversible atrophy along with proliferation of connective tissue,1–3 disintegration of muscle spindles,4,5 deterioration of intramuscular nerve sheaths,6 and From the School of Biomedical Engineering, the Department of Psychiatry and Behavioural Neurosciences, and the Department of Surgery, Division of Plastic Surgery, McMaster University. Received for publication January 5, 2014; accepted March 27, 2014. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000599
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muscle fiber necrosis.7 These structural changes result in decreased muscle force8,9 and motor function and contribute to incomplete or inappropriate muscle reinnervation.6 Considered together, these factors contribute to poor functional recovery following peripheral nerve injury. The criterion standard for treatment of these injuries is immediate nerve repair within 2 months of sustaining an injury.10 However, if the injury site is distant from the muscle, then, regardless of when the initial operation was performed, functional Disclosure: None of the authors has a financial interest in any of the products or devices mentioned in this article.
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Volume 134, Number 5 • Sensory Protection and Muscle Stimulation recovery and outcomes will be poor, as the target musculature will be denervated for long periods during regeneration.6,11 Electrical muscle stimulation has been traditionally used to minimize muscle atrophy and delay denervation-associated changes. However, the efficacy of this approach is controversial, with various studies demonstrating positive and negative effects.12–16 This is partly because of uncertainty regarding optimal stimulation paradigms, different animal models used, and a lack of standardization in animal studies. Thus, electrical stimulation has not been widely adopted as a clinical therapy.17 We and others have shown that a 1-hour/ day regimen of stimulation, easily translated to a clinical setting, can effectively increase muscle mass and force.18,19 However, in these reports, outcome measures following long-term stimulation of denervated muscle were not nearly those of immediately repaired or fully innervated animals, providing evidence that electrical stimulation alone may not fully recover function. Another method used to prevent muscle atrophy and enhance recovery during nerve regeneration is to temporarily suture a sensory nerve to the distal motor nerve stump. This surgical intervention relies on readily available sensory nerves, and in our previous studies we demonstrated that this neurorrhaphy can protect the muscle from denervation-associated atrophy and improve functional recovery.3,5,9,20 This method, called sensory protection, has been successfully used clinically.21 Although sensory protection provides significant benefits, it does not on its own completely prevent denervation-associated changes in muscle. To the best of our knowledge, there have been no studies investigating the combination of sensory protection and chronic electrical muscle stimulation. Sensory protection of denervated muscle maintains structure3,20,22–24 and neurotrophic factor levels25 in the distal tibial stump closer to those of a normal muscle while innervating and protecting muscle spindles that are essential for proprioceptive function.5 Although the sensory nerve does not drive muscle contractions, previous work showed that muscle morphology was improved compared with denervation alone.9 In contrast, electrical muscle stimulation involves contraction of muscle fibers, which has been shown to bulk muscle fibers, leading to concomitant increases in force.13,18,19,26 Others have also shown that muscle excitability is increased27 and that stimulation promotes selective reinnervation.28 This suggests that the two treatments may work by different
mechanisms and may exhibit cooperative effects. We set out to investigate whether the combination of the two therapeutic modalities would improve functional recovery more than each of these alone.
MATERIALS AND METHODS Surgical Procedure All experiments were performed on male Lewis rats (weight, 175 to 200 g) and approved by the Animal Care Committee at McMaster University according to Canadian Council for Animal Care Guidelines. Thirty-nine rats were randomly divided into four experimental groups (n = 8 to 11 per group): denervated, denervated with electrical stimulation, denervated and sensory protected, and sensory protected with electrical stimulation. Details of the surgical procedures for muscle denervation and sensory protection have been described previously elsewhere9 and are also briefly outlined in Figure 1. For muscle stimulation, insulated stainless steel electrodes (AS 631; Cooner Wire, Chatsworth, Calif.) were inserted into the right gastrocnemius muscle of all animals and externalized at the nape to allow connection to the external stimulator.19 One electrode was placed near the motor point in the belly of the muscle and the second electrode was placed distally, closer to the calcaneal tendon. After 3 months of denervation with or without stimulation, sensory protection, or sensory protection with stimulation, all animals underwent motor nerve repair, which involved transecting the common peroneal nerve and suturing it to the distal tibial stump.9 A further 3-month recovery period followed common peroneal nerve repair, for a total of 6 months following initial denervation, before final assessment. Electrical Stimulation Animals in the electrical stimulation group underwent a daily 1-hour session of stimulation 5 days per week delivered using a custom-designed stimulator.19 This paradigm was used throughout the treatment portion of the study (3 months) and for 1 month after common peroneal repair. Each stimulation session featured 600 contractions (one every 6 seconds) using a pulse train of 400-msec duration at a frequency of 100 Hz. These parameters were chosen based on muscle fiber type characteristics and previous work optimizing number of contractions and stimulation time per day.13,18,19 To ensure that a strong contraction was
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Fig. 1. Surgical procedures. The right gastrocnemius muscle in each animal was either denervated (above) or sensory protected (below). Complete denervation was obtained by transection of the tibial nerve 13 mm from the entry to the muscle. The proximal end of the nerve was sutured into the biceps femoris to prevent ectopic innervation. Animals in the denervated groups (denervated, and denervated with electrical stimulation) had the distal tibial stumps ligated, and no further surgical procedures were performed until the delayed nerve repair. In sensory protected animals (denervated and sensory protected, and sensory protected with electrical stimulation) the saphenous nerve was transected and sutured to the distal tibial stump. Each group also had electrodes implanted into the muscle. Animals that were in the stimulated group underwent daily electrical stimulation during the initial 3 months of denervation or sensory protection followed by 1 month of stimulation immediately after surgical nerve repair. The contralateral unoperated limb in each animal served as a control.
elicited, the amplitude was adjusted at each daily session until a visibly strong contraction was established. Others have also used a visual method to assess the strength of the contraction.14,18,29,30 Motor Unit Number Estimation Estimation of the number of motor units was carried out on the experimental right limb in each animal 6 months after the initial denervation. Motor unit numbers were estimated using electromyography and a modified incremental technique using custom-made classification software.31 Force Measurements Once the number of motor units was estimated, the calcaneal tendon was severed and attached to a force transducer, and muscle length was adjusted for maximum force production. Two needle electrodes were placed in the belly of the muscle and served as stimulating electrodes. These were then connected to an isolated stimulator, and maximum
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responses were elicited using a 1-msec stimulus pulse. For tetanic force measurements, a 100-Hz train was delivered for 400 msec. From the twitch force curves, contraction and half-relaxation times were measured. The procedure was repeated for the contralateral limb in all animals. Muscle Weight When all functional measurements were completed, animals were killed by overdose of isoflurane gas, and gastrocnemius muscles were excised and weighed. A section from the mid belly of the medial gastrocnemius muscle was then dissected, mounted in embedding medium, and frozen immediately in liquid nitrogen–cooled isopentane. Analysis of Fast- (Type II) and Slow-Twitch (Type I) Muscle Fibers Muscle samples were sectioned (10 to 12 μm thick) on a cryostat cooled to −20°C (Leica Microsystems, Inc., Buffalo Grove, Ill.) and stained for
Volume 134, Number 5 • Sensory Protection and Muscle Stimulation myofibrillar adenosine triphosphatase at a pH of 10.32 Slides were imaged using a Nikon D300 camera (Nikon Corp., Tokyo, Japan) adapted to a Zeiss Universal light microscope (Carl Zeiss, Oberkochen, Germany). Muscle fiber area and type ratios were analyzed by counting and measuring approximately 2000 fibers (mean ± SD, 2017 ± 632) per animal using ImageJ software (National Institutes of Health, Bethesda, Md.). To visualize fiber area distributions, a smoothed histogram was calculated using a kernel density estimator33 based in Matlab (The MathWorks, Inc., Natick, Mass.). Statistical Analysis To reduce variability resulting from differing animal weights, the ratio of experimental to contralateral limb was used when expressing results for muscle weight and force (twitch and tetanic). A one-way analysis of variance and Tukey post hoc method for multiple comparisons was used to evaluate differences between groups. Histologic data were analyzed using a nonparametric Kruskal-Wallis test with the Dunn post hoc method. Differences were considered significant for values of p < 0.05.
RESULTS Muscle Weights Denervation without any treatment (29 ± 7.5 percent) showed the lowest muscle weight, whereas
the sensory protected with electrical stimulation group had significantly higher weights than all other groups (p < 0.05) (Fig. 2, left).9,34 Although the combined treatment was higher than either denervation with electrical stimulation (41 ± 10.3 percent) or sensory protection (41 ± 7.2 percent) alone, the mean value (53 ± 7.4 percent) was not additive, suggesting that the treatments may work through both common and different mechanisms. There were no differences in muscle weights between denervation with electrical stimulation alone and sensory protection alone. Regardless of the treatment type, mean weights were significantly greater than in the denervation-alone group (p < 0.05) (Fig. 2, left). Motor Unit Number Estimation As expected, denervated animals showed the lowest number of motor units (36 ± 17) (Fig. 2, right). Stimulation during 3 months of denervation and for 1 month after common peroneal nerve repair significantly increased motor unit numbers compared with denervation alone (p < 0.05) (73 ± 32). Although 3 months of sensory protection did not produce a statistically significantly greater number of motor units, the mean value was trending higher compared with denervation (p = 0.097) (63 ± 14). The combination of sensory protection and electrical muscle stimulation produced motor unit counts (92 ± 24) no different than electrical stimulation alone but significantly higher than either
Fig. 2. Muscle weights and motor unit number estimation. (Left) Muscles were excised and weighed 3 months after nerve repair, and weights are expressed as a percentage of the contralateral control limb. The combination treatment of sensory protection and electrical stimulation (SP + ES) was significantly greater than all other groups. Stimulation (ES) alone was no different than sensory protection (SP) alone, and both were greater than denervated (DEN) muscle. (Right) Motor unit (MU) numbers were also estimated 3 months after nerve repair. In each animal, 18 to 20 unique responses were used to estimate the total number of units. The combined treatment of sensory protection and stimulation (SP + ES) was significantly greater than sensory protection (SP) alone or denervation (DEN). Stimulation alone (ES) was significantly greater than denervation. Error bars = SD (one-way analysis of variance followed by Tukey post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001).
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Plastic and Reconstructive Surgery • November 2014 denervation or sensory protection alone (p < 0.001 and p < 0.05, respectively) (Fig. 2, right). These results show that reducing muscle atrophy following denervation is accompanied by an increase in the number of reinnervated motor units. Muscle Forces and Contractile Properties Tetanic tension measurements were elicited using 40 pulses at 100 Hz. Results showed that denervated animals had significantly lower peak tension values (9 ± 7.4 percent) compared with all other groups (p < 0.01) (Fig. 3, above, left).
Electrical stimulation significantly increased tetanic force compared with denervation (29 ± 12.4 percent; p < 0.001). Similarly, sensory protection also significantly increased tetanic force (25 ± 5.7 percent; p < 0.01). Sensory protection with electrical stimulation (38 ± 9.7 percent) exhibited increased tetanic force compared to denervated alone and was significantly greater than sensory protection alone (p < 0.01). There were no observed differences between sensory protection with electrical stimulation and electrical stimulation–alone groups.
Fig. 3. Muscle tension. (Above, left) Mean tetanic force values are represented as a percentage of force values of the contralateral control limbs. For each animal, 40 pulses at 100 Hz were used to obtain tetanic contractions. Stimulation alone (ES) was not different from the combined treatment (SP + ES), but tetanic force of the sensory protected with electrical stimulation group was significantly greater than that of the sensory protected group (SP). All groups had significantly greater force values than the denervated group (DEN). (Above, right) Mean twitch force values are represented as a percentage of force values of the contralateral control limbs. Forces were significantly greater in the combined therapy (sensory protection with electrical stimulation) compared with stimulation alone or denervation. Sensory protected muscle was not significantly different from electrical stimulation alone or sensory protection with electrical stimulation. (Below) Individual twitch profiles for each group. Twitch characteristics such as contraction time and half-relaxation time were not different between groups. Error bars = SD (one-way analysis of variance followed by Tukey post hoc comparison, *p < 0.05, ** p< 0.01, ***p < 0.001).
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Volume 134, Number 5 • Sensory Protection and Muscle Stimulation Twitch forces, expressed as a percentage of the contralateral limb force, followed a similar pattern as muscle weights, with the denervated group (8 ± 3.6 percent) having significantly lower values than all other groups (p < 0.001) (Fig. 3, above, right). Similarly, twitch forces for electrical stimulation (26 ± 10.3 percent) and sensory protection (28 ± 5.6 percent) alone were not different from each other. However, the combination treatment (sensory protection with electrical stimulation, 37 ± 10.7 percent) produced significantly greater twitch forces than electrical stimulation alone (p < 0.05), and mean values were trending higher than sensory protection alone (p = 0.07). Typical tension profiles for each group are shown in Figure 3, below. It is evident from this graph that contractile properties in the muscle remain unaltered. Indeed, there were no statistically significant differences between groups observed in
either contraction or half-relaxation times, suggesting that regardless of the treatment, overall fiber type composition was not altered. Muscle Fiber Type Distribution and Fiber Area To verify that fiber type was unaltered by these treatments, analyses of muscle fiber area and type distribution were assessed in four animals per group, with approximately 2000 fibers examined per animal. Gross histologic examination showed that, although denervated muscle showed large areas of atrophy and fiber necrosis, sensory protection or electrical stimulation alone minimized the number of denervated fascicles (Fig. 4). Muscle sections from animals treated with sensory protection or electrical stimulation exhibited areas of atrophy, suggesting that reinnervation did not take place in some areas. Mean type I muscle fiber areas in electrical stimulation animals were
Fig. 4. Micrographs of adenosine triphosphatase–stained gastrocnemius muscle. Denervated (above, left), stimulated (above, center), sensory protected (above, right), sensory protected and electrically stimulated (below, left) and nonoperated control (below, right) sections. Darkly stained fibers represent type II fast twitch fibers and lightly stained fibers represent type I slow twitch muscle fibers. Electrical stimulation, sensory protection, or the combination of both increases fiber area compared with denervated muscle. Large areas of atrophy are noticeable in all groups except in the combined treatment and nonoperated control. Scale bar = 200 μm.
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Plastic and Reconstructive Surgery • November 2014 significantly larger than fiber areas in denervated animals (p < 0.05) (Table 1). Similarly, electrical stimulation animals had significantly larger type II fiber areas compared with denervated animals (p < 0.01) (Table 1). Type I and type II fiber areas in the sensory protection–alone and sensory protection with electrical stimulation groups were not significantly different from all other groups and were intermediate between denervated and electrical stimulation groups. This suggests that electrical stimulation alone has more impact on fiber area than sensory protection alone. However, the fiber area distributions showed changes that are not reflected in mean area values. A type I fiber distribution (Fig. 5, above) clearly showed that in the denervated group, type I muscle fibers with areas under 1000 μm2 predominated. Stimulated animals had a bimodal distribution of type I fibers, with many more fibers exhibiting larger areas compared with the other groups, suggesting that electrical stimulation alone targets these fibers. Results from our previous studies also showed that our stimulation protocol may target type I fibers.19,34 A type II fiber density plot (Fig. 5, below) shows that all groups except the sensory protection with electrical stimulation group had bimodal distributions. This is consistent with gross histologic examination showing atrophied fascicles in these groups. The sensory protection with electrical stimulation group had a more Gaussian distribution of type II fibers, consistent with the general appearance of the muscle where fascicle atrophy was minimal. The fiber type composition of the muscle was no different between groups, with approximately 10 percent of the counted fibers stained as type I in all groups. Mean fiber area and type composition are shown in Table 1.
DISCUSSION We demonstrate here that the combination of sensory protection and electrical stimulation provides additional benefits beyond either treatment alone. Our results support the hypothesis that
these treatments work by different mechanisms. It was previously reported by Dow et al. that electrical stimulation alone has no effect on functional recovery following long-term denervation and subsequent nerve repair.30 Our work contradicts that study, with 3 months of stimulation providing significantly greater values in all our endpoint tests compared with the denervated group. An important difference between the studies is that we used a freshly axotomized nerve for motor repair, whereas Dow et al. did not. It is known that long-term axotomy can lead to poor functional recovery partly because of nerve degeneration.35 Also, our stimulus paradigm used 1 hour of daily contractions, whereas Dow et al. provided continuous stimulation over a 24-hour period. There is evidence that continuous stimulation may negatively alter muscle reinnervation.36 Nevertheless, our results confirm that intramuscular stimulation alone can improve reinnervation and functional recovery. When electrical stimulation was combined with sensory protection, mean motor unit counts for the sensory protection with electrical stimulation group were larger than for either treatment alone, but not significantly greater than for electrical stimulation alone. When compared with our previous results,34 motor unit values from the sensory protection with electrical stimulation treatment were statistically no different (p > 0.05) from an animal that underwent immediate repair (92 ± 24 versus 82 ± 19, respectively), with immediate repair being the criterion standard exhibiting the best functional recovery following peripheral nerve transection. The results from this functional assay suggest that both treatments improve reinnervation but that electrical stimulation has a greater role. Histologic examination of the muscle showed that the sensory protection with electrical stimulation group had a more uniform-appearing muscle, with intact fascicles exhibiting less atrophy compared with all other groups, confirming improved reinnervation in this group. Fiber type compositions were no different between groups;
Table 1. Rat Gastrocnemius Muscle Fiber Area and Type Distribution after Various Treatments Measurement No. per group Type I area, μm2 Type II area, μm2 Percentage of type I Percentage of type II
DEN
ES
SP
SP + ES
4 301 ± 376 569 ± 517 6.6 ± 2.7 93.4 ± 2.7
4 2055 ± 653* 2224 ± 656* 12.5 ± 5.2 87.5 ± 5.2
4 1054 ± 245 1196 ± 125 10.6 ± 3.1 89.4 ± 3.1
4 1109 ± 344 1648 ± 435 11.7 ± 8.5 88.3 ± 8.5
DEN, denervated; ES, electrical stimulation; SP, sensory protection; SP + ES, sensory protection and electrical stimulation. *Significant difference (p < 0.05) from the denervated group.
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Fig. 5. Fiber area distribution. Distributions of fiber areas for each fiber type are represented as density graphs. (Above) Type I fibers; (below) type II fibers. The peak of the denervated (DEN) group is not shown for scaling purposes. For type I fibers, electrical stimulation alone (ES) had a greater proportion of larger fibers compared with other groups. For type II fibers, both electrical stimulation and sensory protection alone (SP) had bimodal distributions, whereas the distribution of fiber areas in the sensory protection with electrical stimulation group (SP + ES) was uniform in appearance.
however, a bimodal distribution of both type I and type II fibers was present in animals with electrical stimulation alone. Microscopic examination showed that the areas of atrophy were concentrated in the periphery of the muscle sample, suggesting that the stimulation field may not have spread to the entire muscle. Similarly, a bimodal distribution was found for type II fibers in animals with sensory protection alone. The sensory protection with electrical stimulation group did not exhibit these characteristics, but rather showed a decrease in the number of larger type II muscle
fibers compared with the electrical stimulation group. This suggests that sensory protection may limit the increase in fiber size provided by electrical stimulation. In contrast, further investigation into the stimulus amplitudes used showed that sensory protection with electrical stimulation animals had lower amplitudes delivered during each session compared with animals receiving electrical stimulation alone. Contractions were visibly strong in both cases; however, discomfort was more evident at higher amplitudes in those receiving the combined treatment. As the saphenous nerve is a
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Plastic and Reconstructive Surgery • November 2014 sensory cutaneous nerve, electrical stimulation of the muscle during the time that it is sensory protected may depolarize the sensory axons, which may be perceived as cutaneous pain, proprioception, temperature changes, or pressure. Indeed, we have shown that clinical use of sensory protection returns the sense of proprioception.21 This limited use of higher amplitudes in these animal studies results in lower drive to the muscle and may reduce muscle fiber size, limiting therapeutic gain in animals receiving the combined treatment. Animals receiving electrical stimulation alone had higher amplitudes delivered, resulting in greater maintenance of larger fibers. Indeed, the fiber area distribution of electrical stimulation animals was similar to that of animals that underwent immediate repair.34 Stimulation paradigms that lower the amplitude requirements37 may warrant investigation to further enhance functional outcome measures. Other functional outcome measures such as muscle weight and twitch force were significantly higher in the sensory protection with electrical stimulation group compared with either treatment alone. However, outcome measures from combined sensory protection and electrical stimulation alone were not additive. Each outcome measurement was influenced differently by individual treatments. Electrical stimulation had a greater role in producing larger tetanic forces in the combined treatment. This is not surprising, as the contractile mechanism is not activated in sensory protection animals. Both electrical stimulation and sensory protection seemed to equally influence twitch force in animals that received the combined treatment. Muscle weight in the sensory protection with electrical stimulation group was significantly higher than with either treatment alone, suggesting that weight is influenced by both treatments in different ways. The partial additivity of these results may be attributable to the use of lower stimulus amplitudes in the sensory protection with electrical stimulation group. Our previous work supports the theory that these treatments may work through both common and different mechanisms. Sensory protection maintains the structure of the distal nerve stump,3 the proprioceptive machinery,5 and morphologic features without contraction of the muscle fiber. Electrical stimulation further enhances muscle morphology through muscle fiber contraction and improves muscle receptivity to reinnervation. As the distal stump has been shown to play an important role in functional recovery3 and neurotrophin secretion following denervation,25 maintenance of this
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structure may be important to enhanced recovery.38 Muscle atrophy is also an important contributor to poor functional outcomes following nerve injury, and the use of electrical muscle stimulation has been widely shown to reduce atrophy.18,34 Considered together, protection of the distal stump and maintenance of muscle receptivity may provide enhanced functional recovery. The molecular and cellular mechanisms of sensory protection and electrical stimulation are complementary and warrant further investigation.
CONCLUSIONS We provide evidence that sensory protection and electrical muscle stimulation enhance functional recovery to a greater extent than either treatment alone, and that these treatments act at least partially through different mechanisms. Our data support the use of both treatments together to reduce muscle atrophy and enhance functional recovery following denervation, and provide a new strategy for clinical treatment following peripheral nerve injury. Margaret Fahnestock, Ph.D. Department of Psychiatry and Behavioural Neurosciences McMaster University 1280 Main Street West Hamilton, Ontario L8S 4K1, Canada [email protected]
ACKNOWLEDGMENTS
This work was supported in part by grants IMH87057 and CPG-99371 from the Canadian Institutes of Health Research. The authors thank Mary Susan Thompson for preparation of histologic slides and Bhairavi Sivasubramaniam for help with histologic analysis. REFERENCES 1. Gutmann E, Young JZ. The re-innervation of muscle after various periods of atrophy. J Anat. 1944;78:15–43. 2. Savolainen J, Myllylä V, Myllylä R, Vihko V, Väänänen K, Takala TE. Effects of denervation and immobilization on collagen synthesis in rat skeletal muscle and tendon. Am J Physiol. 1988;254:R897–R902. 3. Veltri K, Kwiecien JM, Minet W, Fahnestock M, Bain JR. Contribution of the distal nerve sheath to nerve and muscle preservation following denervation and sensory protection. J Reconstr Microsurg. 2005;21:57–70; discussion 71. 4. Swash M, Fox KP. The pathology of the human muscle spindle: Effect of denervation. J Neurol Sci. 1974;22:1–24. 5. Elsohemy A, Butler R, Bain JR, Fahnestock M. Sensory protection of rat muscle spindles following peripheral nerve injury and reinnervation. Plast Reconstr Surg. 2009;124:1860–1868. 6. Fu SY, Gordon T. Contributing factors to poor functional recovery after delayed nerve repair: Prolonged denervation. J Neurosci. 1995;15:3886–3895.
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23. Wang H, Gu Y, Xu J, Shen L, Li J. Comparative study of different surgical procedures using sensory nerves or neurons for delaying atrophy of denervated skeletal muscle. J Hand Surg Am. 2001;26:326–331. 24. Papakonstantinou KC, Kamin E, Terzis JK. Muscle preservation by prolonged sensory protection. J Reconstr Microsurg. 2002;18:173–182; discussion 183–184. 25. Michalski B, Bain JR, Fahnestock M. Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve. J Neurochem. 2008;105:1244–1252. 26. Kern H, Salmons S, Mayr W, Rossini K, Carraro U. Recovery of long-term denervated human muscles induced by electrical stimulation. Muscle Nerve 2005;31:98–101. 27. Kern H, Hofer C, Mödlin M, Forstner C, Mayr W, Richter W. Functional electrical stimulation (FES) of long-term denervated muscles in humans: Clinical observations and laboratory findings. Basic Appl Myol. 2002;12:291–299. 28. Zealear DL, Rodriguez RJ, Kenny T, et al. Electrical stimulation of a denervated muscle promotes selective reinnervation by native over foreign motoneurons. J Neurophysiol. 2002;87:2195–2199. 29. Dennis RG, Dow DE, Faulkner JA. An implantable device for stimulation of denervated muscles in rats. Med Eng Phys. 2003;25:239–253. 30. Dow DE, Cederna PS, Hassett CA, Dennis RG, Faulkner JA. Electrical stimulation prior to delayed reinnervation does not enhance recovery in muscles of rats. Restor Neurol Neurosci. 2007;25:601–610. 31. Salvador J, de Bruin H. The use of the wavelet transform in EMG M-wave pattern classification. Conf Proc IEEE Eng Med Biol Soc. 2006;1:2304–2307. 32. Brooke MH, Kaiser KK. Muscle fiber types: How many and what kind? Arch Neurol. 1970;23:369–379. 33. Lexell J, Taylor C. “Smoothed histograms”: A visual aid for the analysis of distributions of muscle fiber areas. Muscle Nerve 1991;14:826–828. 34. Willand MP, Holmes M, Bain JR, Fahnestock M, De Bruin H. Electrical muscle stimulation after immediate nerve repair reduces muscle atrophy without affecting reinnervation. Muscle Nerve 2013;48:219–225. 35. Fu SY, Gordon T. Contributing factors to poor functional recovery after delayed nerve repair: Prolonged axotomy. J Neurosci. 1995;15:3876–3885. 36. Love FM, Son YJ, Thompson WJ. Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites. J Neurobiol. 2003;54:566–576. 37. Willand MP, de Bruin H. Design and testing of an instrumentation system to reduce stimulus pulse amplitude requirements during FES. Conf Proc IEEE Eng Med Biol Soc. 2008;2008:2764–2767. 38. Gordon T, Tyreman N, Raji MA. The basis for diminished functional recovery after delayed peripheral nerve repair. J Neurosci. 2011;31:5325–5334.
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Background: Long-term muscle denervation leads to severe and irreversible atrophy coupled with loss of force and motor function. These factors contribute to poor functional recovery following delayed reinnervation. The authors’ previous work demonstrated that temporarily suturing a sensory nerve to the distal motor stump (called sensory protection) significantly reduces muscle atrophy and improves function following reinnervation. The authors have also shown that 1 month of electrical stimulation of denervated muscle significantly improves function and reduces atrophy. In this study, the authors tested whether a combination of sensory protection and electrical stimulation would enhance functional recovery more than either treatment alone. Methods: Rat gastrocnemius muscles were denervated by cutting the tibial nerve. The peroneal nerve was then sutured to the distal tibial stump following 3 months of treatment (i.e., electrical stimulation, sensory protection, or both). Three months after peroneal repair, functional and histologic measurements were taken. Results: All treatment groups had significantly higher muscle weight (p < 0.05) and twitch force (p < 0.001) compared with the untreated group (denervated), but fiber type composition did not differ between groups. Importantly, muscle weight and force were significantly greater in the combined treatment group (p < 0.05) compared with stimulation or sensory protection alone. The combined treatment also produced motor unit counts significantly greater than sensory protection alone (p < 0.05). Conclusions: The combination treatment synergistically reduces atrophy and improves reinnervation and functional measures following delayed nerve repair, suggesting that these approaches work through different mechanisms. The authors’ research supports the clinical use of both modalities together following peripheral nerve injury. (Plast. Reconstr. Surg. 134: 736e, 2014.)
P
eripheral nerve injuries ranging from crush to transection can seriously affect distal muscle structure and function. Longterm denervation is accompanied by irreversible atrophy along with proliferation of connective tissue,1–3 disintegration of muscle spindles,4,5 deterioration of intramuscular nerve sheaths,6 and From the School of Biomedical Engineering, the Department of Psychiatry and Behavioural Neurosciences, and the Department of Surgery, Division of Plastic Surgery, McMaster University. Received for publication January 5, 2014; accepted March 27, 2014. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000599
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muscle fiber necrosis.7 These structural changes result in decreased muscle force8,9 and motor function and contribute to incomplete or inappropriate muscle reinnervation.6 Considered together, these factors contribute to poor functional recovery following peripheral nerve injury. The criterion standard for treatment of these injuries is immediate nerve repair within 2 months of sustaining an injury.10 However, if the injury site is distant from the muscle, then, regardless of when the initial operation was performed, functional Disclosure: None of the authors has a financial interest in any of the products or devices mentioned in this article.
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Volume 134, Number 5 • Sensory Protection and Muscle Stimulation recovery and outcomes will be poor, as the target musculature will be denervated for long periods during regeneration.6,11 Electrical muscle stimulation has been traditionally used to minimize muscle atrophy and delay denervation-associated changes. However, the efficacy of this approach is controversial, with various studies demonstrating positive and negative effects.12–16 This is partly because of uncertainty regarding optimal stimulation paradigms, different animal models used, and a lack of standardization in animal studies. Thus, electrical stimulation has not been widely adopted as a clinical therapy.17 We and others have shown that a 1-hour/ day regimen of stimulation, easily translated to a clinical setting, can effectively increase muscle mass and force.18,19 However, in these reports, outcome measures following long-term stimulation of denervated muscle were not nearly those of immediately repaired or fully innervated animals, providing evidence that electrical stimulation alone may not fully recover function. Another method used to prevent muscle atrophy and enhance recovery during nerve regeneration is to temporarily suture a sensory nerve to the distal motor nerve stump. This surgical intervention relies on readily available sensory nerves, and in our previous studies we demonstrated that this neurorrhaphy can protect the muscle from denervation-associated atrophy and improve functional recovery.3,5,9,20 This method, called sensory protection, has been successfully used clinically.21 Although sensory protection provides significant benefits, it does not on its own completely prevent denervation-associated changes in muscle. To the best of our knowledge, there have been no studies investigating the combination of sensory protection and chronic electrical muscle stimulation. Sensory protection of denervated muscle maintains structure3,20,22–24 and neurotrophic factor levels25 in the distal tibial stump closer to those of a normal muscle while innervating and protecting muscle spindles that are essential for proprioceptive function.5 Although the sensory nerve does not drive muscle contractions, previous work showed that muscle morphology was improved compared with denervation alone.9 In contrast, electrical muscle stimulation involves contraction of muscle fibers, which has been shown to bulk muscle fibers, leading to concomitant increases in force.13,18,19,26 Others have also shown that muscle excitability is increased27 and that stimulation promotes selective reinnervation.28 This suggests that the two treatments may work by different
mechanisms and may exhibit cooperative effects. We set out to investigate whether the combination of the two therapeutic modalities would improve functional recovery more than each of these alone.
MATERIALS AND METHODS Surgical Procedure All experiments were performed on male Lewis rats (weight, 175 to 200 g) and approved by the Animal Care Committee at McMaster University according to Canadian Council for Animal Care Guidelines. Thirty-nine rats were randomly divided into four experimental groups (n = 8 to 11 per group): denervated, denervated with electrical stimulation, denervated and sensory protected, and sensory protected with electrical stimulation. Details of the surgical procedures for muscle denervation and sensory protection have been described previously elsewhere9 and are also briefly outlined in Figure 1. For muscle stimulation, insulated stainless steel electrodes (AS 631; Cooner Wire, Chatsworth, Calif.) were inserted into the right gastrocnemius muscle of all animals and externalized at the nape to allow connection to the external stimulator.19 One electrode was placed near the motor point in the belly of the muscle and the second electrode was placed distally, closer to the calcaneal tendon. After 3 months of denervation with or without stimulation, sensory protection, or sensory protection with stimulation, all animals underwent motor nerve repair, which involved transecting the common peroneal nerve and suturing it to the distal tibial stump.9 A further 3-month recovery period followed common peroneal nerve repair, for a total of 6 months following initial denervation, before final assessment. Electrical Stimulation Animals in the electrical stimulation group underwent a daily 1-hour session of stimulation 5 days per week delivered using a custom-designed stimulator.19 This paradigm was used throughout the treatment portion of the study (3 months) and for 1 month after common peroneal repair. Each stimulation session featured 600 contractions (one every 6 seconds) using a pulse train of 400-msec duration at a frequency of 100 Hz. These parameters were chosen based on muscle fiber type characteristics and previous work optimizing number of contractions and stimulation time per day.13,18,19 To ensure that a strong contraction was
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Fig. 1. Surgical procedures. The right gastrocnemius muscle in each animal was either denervated (above) or sensory protected (below). Complete denervation was obtained by transection of the tibial nerve 13 mm from the entry to the muscle. The proximal end of the nerve was sutured into the biceps femoris to prevent ectopic innervation. Animals in the denervated groups (denervated, and denervated with electrical stimulation) had the distal tibial stumps ligated, and no further surgical procedures were performed until the delayed nerve repair. In sensory protected animals (denervated and sensory protected, and sensory protected with electrical stimulation) the saphenous nerve was transected and sutured to the distal tibial stump. Each group also had electrodes implanted into the muscle. Animals that were in the stimulated group underwent daily electrical stimulation during the initial 3 months of denervation or sensory protection followed by 1 month of stimulation immediately after surgical nerve repair. The contralateral unoperated limb in each animal served as a control.
elicited, the amplitude was adjusted at each daily session until a visibly strong contraction was established. Others have also used a visual method to assess the strength of the contraction.14,18,29,30 Motor Unit Number Estimation Estimation of the number of motor units was carried out on the experimental right limb in each animal 6 months after the initial denervation. Motor unit numbers were estimated using electromyography and a modified incremental technique using custom-made classification software.31 Force Measurements Once the number of motor units was estimated, the calcaneal tendon was severed and attached to a force transducer, and muscle length was adjusted for maximum force production. Two needle electrodes were placed in the belly of the muscle and served as stimulating electrodes. These were then connected to an isolated stimulator, and maximum
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responses were elicited using a 1-msec stimulus pulse. For tetanic force measurements, a 100-Hz train was delivered for 400 msec. From the twitch force curves, contraction and half-relaxation times were measured. The procedure was repeated for the contralateral limb in all animals. Muscle Weight When all functional measurements were completed, animals were killed by overdose of isoflurane gas, and gastrocnemius muscles were excised and weighed. A section from the mid belly of the medial gastrocnemius muscle was then dissected, mounted in embedding medium, and frozen immediately in liquid nitrogen–cooled isopentane. Analysis of Fast- (Type II) and Slow-Twitch (Type I) Muscle Fibers Muscle samples were sectioned (10 to 12 μm thick) on a cryostat cooled to −20°C (Leica Microsystems, Inc., Buffalo Grove, Ill.) and stained for
Volume 134, Number 5 • Sensory Protection and Muscle Stimulation myofibrillar adenosine triphosphatase at a pH of 10.32 Slides were imaged using a Nikon D300 camera (Nikon Corp., Tokyo, Japan) adapted to a Zeiss Universal light microscope (Carl Zeiss, Oberkochen, Germany). Muscle fiber area and type ratios were analyzed by counting and measuring approximately 2000 fibers (mean ± SD, 2017 ± 632) per animal using ImageJ software (National Institutes of Health, Bethesda, Md.). To visualize fiber area distributions, a smoothed histogram was calculated using a kernel density estimator33 based in Matlab (The MathWorks, Inc., Natick, Mass.). Statistical Analysis To reduce variability resulting from differing animal weights, the ratio of experimental to contralateral limb was used when expressing results for muscle weight and force (twitch and tetanic). A one-way analysis of variance and Tukey post hoc method for multiple comparisons was used to evaluate differences between groups. Histologic data were analyzed using a nonparametric Kruskal-Wallis test with the Dunn post hoc method. Differences were considered significant for values of p < 0.05.
RESULTS Muscle Weights Denervation without any treatment (29 ± 7.5 percent) showed the lowest muscle weight, whereas
the sensory protected with electrical stimulation group had significantly higher weights than all other groups (p < 0.05) (Fig. 2, left).9,34 Although the combined treatment was higher than either denervation with electrical stimulation (41 ± 10.3 percent) or sensory protection (41 ± 7.2 percent) alone, the mean value (53 ± 7.4 percent) was not additive, suggesting that the treatments may work through both common and different mechanisms. There were no differences in muscle weights between denervation with electrical stimulation alone and sensory protection alone. Regardless of the treatment type, mean weights were significantly greater than in the denervation-alone group (p < 0.05) (Fig. 2, left). Motor Unit Number Estimation As expected, denervated animals showed the lowest number of motor units (36 ± 17) (Fig. 2, right). Stimulation during 3 months of denervation and for 1 month after common peroneal nerve repair significantly increased motor unit numbers compared with denervation alone (p < 0.05) (73 ± 32). Although 3 months of sensory protection did not produce a statistically significantly greater number of motor units, the mean value was trending higher compared with denervation (p = 0.097) (63 ± 14). The combination of sensory protection and electrical muscle stimulation produced motor unit counts (92 ± 24) no different than electrical stimulation alone but significantly higher than either
Fig. 2. Muscle weights and motor unit number estimation. (Left) Muscles were excised and weighed 3 months after nerve repair, and weights are expressed as a percentage of the contralateral control limb. The combination treatment of sensory protection and electrical stimulation (SP + ES) was significantly greater than all other groups. Stimulation (ES) alone was no different than sensory protection (SP) alone, and both were greater than denervated (DEN) muscle. (Right) Motor unit (MU) numbers were also estimated 3 months after nerve repair. In each animal, 18 to 20 unique responses were used to estimate the total number of units. The combined treatment of sensory protection and stimulation (SP + ES) was significantly greater than sensory protection (SP) alone or denervation (DEN). Stimulation alone (ES) was significantly greater than denervation. Error bars = SD (one-way analysis of variance followed by Tukey post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001).
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Plastic and Reconstructive Surgery • November 2014 denervation or sensory protection alone (p < 0.001 and p < 0.05, respectively) (Fig. 2, right). These results show that reducing muscle atrophy following denervation is accompanied by an increase in the number of reinnervated motor units. Muscle Forces and Contractile Properties Tetanic tension measurements were elicited using 40 pulses at 100 Hz. Results showed that denervated animals had significantly lower peak tension values (9 ± 7.4 percent) compared with all other groups (p < 0.01) (Fig. 3, above, left).
Electrical stimulation significantly increased tetanic force compared with denervation (29 ± 12.4 percent; p < 0.001). Similarly, sensory protection also significantly increased tetanic force (25 ± 5.7 percent; p < 0.01). Sensory protection with electrical stimulation (38 ± 9.7 percent) exhibited increased tetanic force compared to denervated alone and was significantly greater than sensory protection alone (p < 0.01). There were no observed differences between sensory protection with electrical stimulation and electrical stimulation–alone groups.
Fig. 3. Muscle tension. (Above, left) Mean tetanic force values are represented as a percentage of force values of the contralateral control limbs. For each animal, 40 pulses at 100 Hz were used to obtain tetanic contractions. Stimulation alone (ES) was not different from the combined treatment (SP + ES), but tetanic force of the sensory protected with electrical stimulation group was significantly greater than that of the sensory protected group (SP). All groups had significantly greater force values than the denervated group (DEN). (Above, right) Mean twitch force values are represented as a percentage of force values of the contralateral control limbs. Forces were significantly greater in the combined therapy (sensory protection with electrical stimulation) compared with stimulation alone or denervation. Sensory protected muscle was not significantly different from electrical stimulation alone or sensory protection with electrical stimulation. (Below) Individual twitch profiles for each group. Twitch characteristics such as contraction time and half-relaxation time were not different between groups. Error bars = SD (one-way analysis of variance followed by Tukey post hoc comparison, *p < 0.05, ** p< 0.01, ***p < 0.001).
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Volume 134, Number 5 • Sensory Protection and Muscle Stimulation Twitch forces, expressed as a percentage of the contralateral limb force, followed a similar pattern as muscle weights, with the denervated group (8 ± 3.6 percent) having significantly lower values than all other groups (p < 0.001) (Fig. 3, above, right). Similarly, twitch forces for electrical stimulation (26 ± 10.3 percent) and sensory protection (28 ± 5.6 percent) alone were not different from each other. However, the combination treatment (sensory protection with electrical stimulation, 37 ± 10.7 percent) produced significantly greater twitch forces than electrical stimulation alone (p < 0.05), and mean values were trending higher than sensory protection alone (p = 0.07). Typical tension profiles for each group are shown in Figure 3, below. It is evident from this graph that contractile properties in the muscle remain unaltered. Indeed, there were no statistically significant differences between groups observed in
either contraction or half-relaxation times, suggesting that regardless of the treatment, overall fiber type composition was not altered. Muscle Fiber Type Distribution and Fiber Area To verify that fiber type was unaltered by these treatments, analyses of muscle fiber area and type distribution were assessed in four animals per group, with approximately 2000 fibers examined per animal. Gross histologic examination showed that, although denervated muscle showed large areas of atrophy and fiber necrosis, sensory protection or electrical stimulation alone minimized the number of denervated fascicles (Fig. 4). Muscle sections from animals treated with sensory protection or electrical stimulation exhibited areas of atrophy, suggesting that reinnervation did not take place in some areas. Mean type I muscle fiber areas in electrical stimulation animals were
Fig. 4. Micrographs of adenosine triphosphatase–stained gastrocnemius muscle. Denervated (above, left), stimulated (above, center), sensory protected (above, right), sensory protected and electrically stimulated (below, left) and nonoperated control (below, right) sections. Darkly stained fibers represent type II fast twitch fibers and lightly stained fibers represent type I slow twitch muscle fibers. Electrical stimulation, sensory protection, or the combination of both increases fiber area compared with denervated muscle. Large areas of atrophy are noticeable in all groups except in the combined treatment and nonoperated control. Scale bar = 200 μm.
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Plastic and Reconstructive Surgery • November 2014 significantly larger than fiber areas in denervated animals (p < 0.05) (Table 1). Similarly, electrical stimulation animals had significantly larger type II fiber areas compared with denervated animals (p < 0.01) (Table 1). Type I and type II fiber areas in the sensory protection–alone and sensory protection with electrical stimulation groups were not significantly different from all other groups and were intermediate between denervated and electrical stimulation groups. This suggests that electrical stimulation alone has more impact on fiber area than sensory protection alone. However, the fiber area distributions showed changes that are not reflected in mean area values. A type I fiber distribution (Fig. 5, above) clearly showed that in the denervated group, type I muscle fibers with areas under 1000 μm2 predominated. Stimulated animals had a bimodal distribution of type I fibers, with many more fibers exhibiting larger areas compared with the other groups, suggesting that electrical stimulation alone targets these fibers. Results from our previous studies also showed that our stimulation protocol may target type I fibers.19,34 A type II fiber density plot (Fig. 5, below) shows that all groups except the sensory protection with electrical stimulation group had bimodal distributions. This is consistent with gross histologic examination showing atrophied fascicles in these groups. The sensory protection with electrical stimulation group had a more Gaussian distribution of type II fibers, consistent with the general appearance of the muscle where fascicle atrophy was minimal. The fiber type composition of the muscle was no different between groups, with approximately 10 percent of the counted fibers stained as type I in all groups. Mean fiber area and type composition are shown in Table 1.
DISCUSSION We demonstrate here that the combination of sensory protection and electrical stimulation provides additional benefits beyond either treatment alone. Our results support the hypothesis that
these treatments work by different mechanisms. It was previously reported by Dow et al. that electrical stimulation alone has no effect on functional recovery following long-term denervation and subsequent nerve repair.30 Our work contradicts that study, with 3 months of stimulation providing significantly greater values in all our endpoint tests compared with the denervated group. An important difference between the studies is that we used a freshly axotomized nerve for motor repair, whereas Dow et al. did not. It is known that long-term axotomy can lead to poor functional recovery partly because of nerve degeneration.35 Also, our stimulus paradigm used 1 hour of daily contractions, whereas Dow et al. provided continuous stimulation over a 24-hour period. There is evidence that continuous stimulation may negatively alter muscle reinnervation.36 Nevertheless, our results confirm that intramuscular stimulation alone can improve reinnervation and functional recovery. When electrical stimulation was combined with sensory protection, mean motor unit counts for the sensory protection with electrical stimulation group were larger than for either treatment alone, but not significantly greater than for electrical stimulation alone. When compared with our previous results,34 motor unit values from the sensory protection with electrical stimulation treatment were statistically no different (p > 0.05) from an animal that underwent immediate repair (92 ± 24 versus 82 ± 19, respectively), with immediate repair being the criterion standard exhibiting the best functional recovery following peripheral nerve transection. The results from this functional assay suggest that both treatments improve reinnervation but that electrical stimulation has a greater role. Histologic examination of the muscle showed that the sensory protection with electrical stimulation group had a more uniform-appearing muscle, with intact fascicles exhibiting less atrophy compared with all other groups, confirming improved reinnervation in this group. Fiber type compositions were no different between groups;
Table 1. Rat Gastrocnemius Muscle Fiber Area and Type Distribution after Various Treatments Measurement No. per group Type I area, μm2 Type II area, μm2 Percentage of type I Percentage of type II
DEN
ES
SP
SP + ES
4 301 ± 376 569 ± 517 6.6 ± 2.7 93.4 ± 2.7
4 2055 ± 653* 2224 ± 656* 12.5 ± 5.2 87.5 ± 5.2
4 1054 ± 245 1196 ± 125 10.6 ± 3.1 89.4 ± 3.1
4 1109 ± 344 1648 ± 435 11.7 ± 8.5 88.3 ± 8.5
DEN, denervated; ES, electrical stimulation; SP, sensory protection; SP + ES, sensory protection and electrical stimulation. *Significant difference (p < 0.05) from the denervated group.
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Fig. 5. Fiber area distribution. Distributions of fiber areas for each fiber type are represented as density graphs. (Above) Type I fibers; (below) type II fibers. The peak of the denervated (DEN) group is not shown for scaling purposes. For type I fibers, electrical stimulation alone (ES) had a greater proportion of larger fibers compared with other groups. For type II fibers, both electrical stimulation and sensory protection alone (SP) had bimodal distributions, whereas the distribution of fiber areas in the sensory protection with electrical stimulation group (SP + ES) was uniform in appearance.
however, a bimodal distribution of both type I and type II fibers was present in animals with electrical stimulation alone. Microscopic examination showed that the areas of atrophy were concentrated in the periphery of the muscle sample, suggesting that the stimulation field may not have spread to the entire muscle. Similarly, a bimodal distribution was found for type II fibers in animals with sensory protection alone. The sensory protection with electrical stimulation group did not exhibit these characteristics, but rather showed a decrease in the number of larger type II muscle
fibers compared with the electrical stimulation group. This suggests that sensory protection may limit the increase in fiber size provided by electrical stimulation. In contrast, further investigation into the stimulus amplitudes used showed that sensory protection with electrical stimulation animals had lower amplitudes delivered during each session compared with animals receiving electrical stimulation alone. Contractions were visibly strong in both cases; however, discomfort was more evident at higher amplitudes in those receiving the combined treatment. As the saphenous nerve is a
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Plastic and Reconstructive Surgery • November 2014 sensory cutaneous nerve, electrical stimulation of the muscle during the time that it is sensory protected may depolarize the sensory axons, which may be perceived as cutaneous pain, proprioception, temperature changes, or pressure. Indeed, we have shown that clinical use of sensory protection returns the sense of proprioception.21 This limited use of higher amplitudes in these animal studies results in lower drive to the muscle and may reduce muscle fiber size, limiting therapeutic gain in animals receiving the combined treatment. Animals receiving electrical stimulation alone had higher amplitudes delivered, resulting in greater maintenance of larger fibers. Indeed, the fiber area distribution of electrical stimulation animals was similar to that of animals that underwent immediate repair.34 Stimulation paradigms that lower the amplitude requirements37 may warrant investigation to further enhance functional outcome measures. Other functional outcome measures such as muscle weight and twitch force were significantly higher in the sensory protection with electrical stimulation group compared with either treatment alone. However, outcome measures from combined sensory protection and electrical stimulation alone were not additive. Each outcome measurement was influenced differently by individual treatments. Electrical stimulation had a greater role in producing larger tetanic forces in the combined treatment. This is not surprising, as the contractile mechanism is not activated in sensory protection animals. Both electrical stimulation and sensory protection seemed to equally influence twitch force in animals that received the combined treatment. Muscle weight in the sensory protection with electrical stimulation group was significantly higher than with either treatment alone, suggesting that weight is influenced by both treatments in different ways. The partial additivity of these results may be attributable to the use of lower stimulus amplitudes in the sensory protection with electrical stimulation group. Our previous work supports the theory that these treatments may work through both common and different mechanisms. Sensory protection maintains the structure of the distal nerve stump,3 the proprioceptive machinery,5 and morphologic features without contraction of the muscle fiber. Electrical stimulation further enhances muscle morphology through muscle fiber contraction and improves muscle receptivity to reinnervation. As the distal stump has been shown to play an important role in functional recovery3 and neurotrophin secretion following denervation,25 maintenance of this
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structure may be important to enhanced recovery.38 Muscle atrophy is also an important contributor to poor functional outcomes following nerve injury, and the use of electrical muscle stimulation has been widely shown to reduce atrophy.18,34 Considered together, protection of the distal stump and maintenance of muscle receptivity may provide enhanced functional recovery. The molecular and cellular mechanisms of sensory protection and electrical stimulation are complementary and warrant further investigation.
CONCLUSIONS We provide evidence that sensory protection and electrical muscle stimulation enhance functional recovery to a greater extent than either treatment alone, and that these treatments act at least partially through different mechanisms. Our data support the use of both treatments together to reduce muscle atrophy and enhance functional recovery following denervation, and provide a new strategy for clinical treatment following peripheral nerve injury. Margaret Fahnestock, Ph.D. Department of Psychiatry and Behavioural Neurosciences McMaster University 1280 Main Street West Hamilton, Ontario L8S 4K1, Canada [email protected]
ACKNOWLEDGMENTS
This work was supported in part by grants IMH87057 and CPG-99371 from the Canadian Institutes of Health Research. The authors thank Mary Susan Thompson for preparation of histologic slides and Bhairavi Sivasubramaniam for help with histologic analysis. REFERENCES 1. Gutmann E, Young JZ. The re-innervation of muscle after various periods of atrophy. J Anat. 1944;78:15–43. 2. Savolainen J, Myllylä V, Myllylä R, Vihko V, Väänänen K, Takala TE. Effects of denervation and immobilization on collagen synthesis in rat skeletal muscle and tendon. Am J Physiol. 1988;254:R897–R902. 3. Veltri K, Kwiecien JM, Minet W, Fahnestock M, Bain JR. Contribution of the distal nerve sheath to nerve and muscle preservation following denervation and sensory protection. J Reconstr Microsurg. 2005;21:57–70; discussion 71. 4. Swash M, Fox KP. The pathology of the human muscle spindle: Effect of denervation. J Neurol Sci. 1974;22:1–24. 5. Elsohemy A, Butler R, Bain JR, Fahnestock M. Sensory protection of rat muscle spindles following peripheral nerve injury and reinnervation. Plast Reconstr Surg. 2009;124:1860–1868. 6. Fu SY, Gordon T. Contributing factors to poor functional recovery after delayed nerve repair: Prolonged denervation. J Neurosci. 1995;15:3886–3895.
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