Early Cyclical Neuromuscular Electrical Stimulation Improves Strength and Trophism by Akt Pathway Signaling in Partially Paralyzed Biceps Muscle After Spinal Cord Injury in Rats

Early Cyclical Neuromuscular Electrical Stimulation Improves Strength and Trophism by Akt Pathway... Abstract Background Electrical stimulation is often used to treat weakness in people with spinal cord injury (SCI); however its efficacy for increasing strength and trophism is weak, and the mechanisms underlying the therapeutic benefits are unknown. Objective The purpose of this study was to analyze the effects of neuromuscular electrical stimulation (NMES) on muscle function, trophism, and the Akt pathway signaling involved in muscular plasticity after incomplete SCI in rats. Design This was an experimental study. Methods Twenty-one adult female Wistar rats were divided into sham, SCI, and SCI plus NMES groups. In injured animals, SCI hemisection was induced by a surgical procedure at the C5-C7 level. The 5-week NMES protocol consisted of biceps brachii muscle stimulation 5 times per week, initiated 48 h after injury. Forepaw function and strength, biceps muscle trophism, and the expression of phosphorylated Akt, p70S6K, and GSK-3ß cellular anabolic pathway markers in stimulated muscle tissue were assessed. Results There was an increase in bicep muscle strength in the NMES group compared with the untreated SCI group, from postoperative day 21 until the end of the evaluation period. Also, there was an increase in muscle trophism in the NMES group compared with the SCI group. Forelimb function gradually recovered in both the SCI group and the NMES group, with no differences between them. Regarding muscle protein expression, the NMES group had higher values for phospho-Akt, phospho-p70S6K, and phospho-GSK-3ß than did the SCI group. Limitations The experimental findings were limited to an animal model of incomplete SCI and may not be fully generalizable to humans. Conclusions Early cyclical NMES therapy was shown to increase muscle strength and induce hypertrophy after incomplete SCI in a rat model, probably by increasing phospho-Akt, phospho-p70S6K, and phospho-GSK-3ß signaling protein synthesis. In individuals with spinal cord injury (SCI), muscle tissue is exposed to unfavorable situations, which culminate in a rapid loss of muscle mass, causing an hypotrophic state, mainly due to muscle disuse.1–3 To treat weakness in partially paralyzed muscles after a SCI, physical therapists frequently use electrical stimulation therapy.4 Despite the routine use of this mode of therapy, the efficacy of neuromuscular electrical stimulation (NMES) in increasing muscle strength and trophism is poorly studied and the mechanisms underlying the therapeutic benefits of NMES are unknown.4,5 Against the scarcity of the studies to prove the efficacy of NMES to improve strength and trophism in partially paralyzed muscle, animal models of incomplete SCI may provide some preclinical behavioral and biological evidence about the beneficial effects of the NMES in this condition. This can supports its clinical application and put forward new clinical studies with a better methodological design. However, while numerous studies have analyzed the motor and functional recovery of the anterior and posterior paws in animal models of SCI6–11 few studies have investigated the effects of NMES in such models.12,13 Beaumont et al performed 5 days of NMES associated with a functional activity (gait; functional NMES; 3 times/day), with electrodes implanted in the quadriceps and hamstring muscles in a model of SCI contusion at the T10 level.12 They found an improvement in locomotion recovery by day 7 post-SCI, measured by Basso-Beattie-Bresnahan scores.12 Another study, by Butezloff et al, in a model of SCI transection at the same level T10, consisted of 4 weeks of NMES not associated with a task, but only muscle contraction by electric stimulation (cyclical NMES; 3 days/week; 20 min/day).13 They found partial muscle hypotrophy prevention in the NMES group, showed by increased mechanical resistance and muscle mass, and a smaller deposit of fibrotic tissue in the muscles that received passive muscle contraction with cyclical NMES treatment.13 However, none of the studies analyzed the recovery of muscle strength or the possible signaling pathways involved in muscle trophism after NMES treatment. In fact, after a SCI, there is an unbalance between the muscle protein turnover that culminates with a severe muscle hypotrophy. A significant reduction of proteins participating in the muscle protein synthesis pathway was found, with 54% reduction in Akt protein, a 48% reduction in phosphorylated mammalian target of rapamycin (mTOR) protein and a 60% reduction in phosphorylated p70S6K protein.1 Surprisingly, this significant muscle loss observed in SCI population was not associated with the increase in the muscle proteolytic pathway.1,14 It is well established that physical exercise with voluntary contraction promotes muscle hypertrophy, with serine/threonine kinase (Akt)/mTOR activation being the main pathway.14–16 The trophic status of the musculature is regulated in response to changes in workload and activity, and an improvement in muscle mass “hypertrophy” is associated with increased protein synthesis.15,17,18 Despite the knowledge of this hypertrophic pathway induced by active exercise, it remains unknown whether the passive contraction induced by cyclical NMES or active-assisted contraction induced by functional NMES promotes activation of the same Akt/mTOR pathway. No studies have examined the effects and mechanisms of cyclical NMES on strength and muscle hypertrophy after SCI. To mimic the clinical condition, a rat model of incomplete SCI with cyclical NMES of partially paralyzed muscles is used to assess the possible beneficial effects of NMES therapy. Specifically, we hypothesize that cyclical NMES improves muscular function and trophism and that this muscular plasticity may be accomplished by cellular mechanisms similar to those involved in active exercises. Methods Spinal Cord Injury Procedures Adult female Wistar rats (180–200 g, n = 21) were randomly divided into 3 groups: control (sham, n = 8), SCI (n = 7), and SCI plus NMES (n = 6). The surgical procedures for preparing and performing C5-C7 SCI hemisection followed previously published methods.19 Briefly, rats were anesthetized via an intraperitoneal injection of a ketamine (87 mg/kg) and xylazine (12 mg/kg) cocktail. Once properly anesthetized, the animals’ necks were shaved and disinfected with 70% ethanol, and then the skin and musculature were carefully dissected to expose the cervical vertebrae. A complete laminectomy of the C5-C7 level was performed to expose the dorsal surface of the spinal cord, and the hemisection injury was inflicted using microscissors on the right side. The muscle and skin were closed in layers. The same surgical procedure without SCI hemisection was carried out in the sham animals. The rats were then placed in a clean home cage in a heat-controlled environment under careful monitoring by the surgeon. Recovered animals had access to food and water ad libitum. The animals received 2.5 mg/kg of enrofloxacin (antibiotic) subcutaneously (Baytril Bayer S.A., São Paulo, Brazil) immediately, and until postoperative day 3. The bladders of injured rats were expressed twice daily until micturition control returned. Neuromuscular Electrical Stimulation Therapy In the NMES group, the electrical stimulation therapy, adapted from Ambrosio et al,20 was conducted using the 2-channel Neurodyn portable device (Ibramed, São Paulo, Brazil) with animals under isoflurane (5%) inhalation anesthesia, beginning 48 hours postinjury. The therapy schedule followed a regimen of 10 min per session, once a day, 5 days per week, with a total therapy time of 5 weeks, as shown in Figure 1A. The anterior part of the right forepaws were shaved for coupling of electrodes on the skin in the biceps brachii muscle region shown in Figure 1B. Electrical stimulation parameters were as follows: biphasic asymmetric pulses; pulse duration of 150 μs; stimulation frequency of 50 Hz; and intensity adjusted to produce visible muscle contraction and progressed over time in order to maintain effective biceps brachii muscle contraction, perceived by visualization of the movement through all of the elbow flexion. Figure 1. View largeDownload slide Flowchart of the experimental time line (A) and image of the electrode placement (B). Figure 1. View largeDownload slide Flowchart of the experimental time line (A) and image of the electrode placement (B). Grip Test A grip strength meter (Columbus Instruments, Columbus, Ohio) was used to measure maximum forelimb grip strength. The meter consisted of a force transducer positioned between a metal bar. Rats were held around the midsection with 1 forelimb restrained and the other forepaw allowed to grasp the bar. The rat was gently pulled away from the device and the maximum force (g) of the grasping was recorded just before the rats release the bar. Rats were acclimated to the grip strength meter (10 trials) 1 week prior to the first testing session. Care was taken to ensure the grip was a voluntary grasp followed by release, rather than a spastic digit contracture. Testing sessions consisted of 3 successive trials, and the forelimb strength was measured once a week, until the end of the protocol.21 Irvine-Beattie-Bresnahan (IBB) Forelimb Recovery Scale To reduce the stress of the exposure to a new environment and to facilitate task learning, animals were trained to eat cereal in the test environment during the 2 weeks prior to surgery. On the test day, the animals were habituated to the laboratory conditions. Afterward, using a digital camera (Sanyo camera, digital 10) they were filmed eating 1 entire doughnut-shaped cereal piece (Kellogg's fruit loops). The animals were exposed to cereal positioned inside a cylinder with acrylic mirrors on the sides. Assessment began when the animal grasped the cereal piece, with the evaluator filming from a distance of 1 m to provide adequate zoom when the animal moved. The images were evaluated using Windows Media Player 10, slowed down to 50% or less. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping techniques.22,23 The data for each piece was collected on the score sheet described by Irvine et al23,24 and the analysis was made with just 1 piece of cereal by animal. Animals were assessed for the most common position (more than 50% of the time), referred to as the predominant position. Following these detailed assessments, the examiner filled in the score sheet for each animal and then gave a score from 0 to 9, where 0 represents the worst functionality and 9 represents normal behavior. This test was performed on baseline and on days 2, 7, 14, 21, 28, and 35 after surgery. The IBB test has strong interrater reliability (experienced raters: 0.16 ± 0.15) and validity (F = 120.89; df = 3,24; P < .00001; η2 = 0.94) to measure forelimb and fine digital control in rats, as shown by Irvine et al.24 Furthermore, the IBB test proved to be highly sensitive to the injury × time interaction (F = 7.20; df = 9,72; P <. 00001; η2 = 0.47).24 Muscle Trophism Assessment Rats were anesthetized via an intraperitoneal injection of a ketamine (87 mg/kg) and xylazine (12 mg/kg) cocktail 24 hours after the last NMES therapy session, weighed, and euthanized. The right biceps brachii muscle was dissected and weighed on a precision scale to calculate the muscle trophism, measured by the ratio of muscle weight/body weight.25 Afterward, samples of the central portion of each muscle were excised under ice, frozen in liquid nitrogen, and stored at −70°C until processed for biochemical analysis. Biochemical Analysis Muscle trophism pathway proteins were measured by an enzyme-linked immunosorbent assay (ELISA). The muscle samples were homogenized in a Ultra-Turrax Homogenizer (T-18; IKA Works, Wilmington, North Carolina) with a phosphate-buffered saline solution containing: 1 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 5 mM sodium fluoride, 6 M urea, leupeptin (10 μg/mL), pepstatin (10 μg/mL), 100 μM phenylmethylsulfonyl fluoride, aprotinin (3 μg/mL), 2.5 mM sodium pyrophosphate, and 1 mM activated sodium orthovanadate. The homogenates were transferred to 1.5-mL Eppendorf tubes and centrifuged at 3000 × g for 10 min at 4°C, and the supernatant obtained was stored at −70°C until further analysis. Total protein content was measured in the supernatant using the method of Bradford.26 The muscle tissue levels of glycogen synthase kinase-3 beta phosphorylated at S9 (phospho-GSK-3β [S9], catalog number DYC1590E), protein kinase B (Akt) phosphorylated at S473 (phospho-Akt [S473], catalog number DYC887B-2), and p70 ribosomal S6 kinase phosphorylated at T421 and S424 (phospho-p70 S6 kinase [T421/S424], catalog number DYC8965-5) were measured using sandwich ELISA according to the manufacturer's instructions (DuoSet ELISA R&D Systems, Minneapolis, Minnesota). The muscle protein levels were estimated by interpolation from a standard curve using colorimetric measurements at 450 nm (correction wavelength = 540 nm) in an ELISA plate reader (Apollo-8 LB 912; Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany). All results were expressed as pg/mg of protein. Data Analysis Comparisons among groups were performed using SPSS 20.0 software. The results of the IBB test and the grip test were analyzed using a 2-way analysis of variance (ANOVA) for repeated measures and the results of the trophism and biochemical analysis using a 1-way ANOVA. Afterward, the Tukey post hoc test for multiple comparisons was carried out. Data showed normal distribution, as analyzed by the Kolmogorov-Smirnov test. Significance levels were set at 5% (P ≤ .05). Values were expressed as the mean ± standard error of mean (SEM) or standard deviation (SD). Role of the Funding Source This study was funded by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (Foundation for Research and Innovation Support of the State of Santa Catarina) (FAPESC) (TO 2014TR2256). Financing was used to purchase ELISA kits and electrical stimulation equipment. Results NMES Accelerates Forelimb Function Recovery After Incomplete SCI Muscle strength recovery The muscle strength recovery was measured with a grip strength meter, at baseline and once a week over the 5-week cyclical NMES protocol period. The ANOVA for repeated measures demonstrated significant main effects for groups (F = 106.2; df = 2,19; P < .001; power = 1.00), for time (F = 43.1; df = 6,114; P < .001; power = 1.00) and for interaction between time and group (F = 6.2; df = 12,114; P < .001; power = 1.00) effects. The results of the groups over the period are shown in Figure 2A. In addition, the mean, standard deviation and difference between groups for injured groups are shown in the Table. Figure 2. View largeDownload slide Line plots showing the mean ± standard error of the mean for grip test muscle strength (A) and Irvine-Beattie-Bresnahan (IBB) test (B) recovery throughout the 5-week neuromuscular electrical stimulation (NMES) protocol. * = significant difference (P < .05) compared with the sham group; # = significant difference (P < .05) compared with the spinal cord injury (SCI) group. Figure 2. View largeDownload slide Line plots showing the mean ± standard error of the mean for grip test muscle strength (A) and Irvine-Beattie-Bresnahan (IBB) test (B) recovery throughout the 5-week neuromuscular electrical stimulation (NMES) protocol. * = significant difference (P < .05) compared with the sham group; # = significant difference (P < .05) compared with the spinal cord injury (SCI) group. Table. Forelimb Grip Strength (Grip Test)a Postoperative Day  Forelimb Grip Strength, g, Mean (SD)  Differences Between Groups  Sham and SCI  Sham and NMES  SCI and NMES  Sham Group  SCI 
Group  NMES Group  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Baseline  115.4 (25.2)  110.5 (12.5)  106.0 (15.5)  4.9 (−20.8 to 30.7)  < .001  9.4 (−16.4 to 35.2)  1.0  4.5 (−22.2 to 31.1)  1.0  2  89.1 (5.4)  3.1 (5.4)  4.14 (5.39)  86.0 (77.3 to 94.6)  < .001  85.0 (76.3 to 93.6)  < .001  −1.0 (−9.9 to 7.9)  1.0  7  121.9 (22.9)  36.9 (13.5)  48.4 (24.7)  85.0 (56.4 to 113.6)  < .001  73.5 (44.8 to 102.1)  < .001  −11.6 (−41.1 to 18.0)  .95  14  127.0 (30.2)  49.4 (7.7)  52.7 (20.7)  77.6 (47.5 to 107.8)  < .001  74.2 (44.1 to 104.4)  < .001  −3.4 (−34.5 to 27.8)  1.0  21  120.3 (14.6)  47.5 (13.4)  87.3 (26.2)  72.8 (47.3 to 98.3)  < .001  32.9 (7.4 to 58.5)  .009  −39.8 (−66.2 to −13.5)  .002  28  141.3 (33.2)  66.3 (15.9)  110.3 (26.6)  75.0 (38.8 to 111.2)  < .001  31.1 (−5.0 to 67.2)  .18  −43.9 (−81.3 to −6.5)  .018  35  149.5 (23.1)  74.6 (32.5)  111.1 (17.8)  74.9 (40.8 to 109.1)  < .001  38.4 (4.3 to 72.5)  .024  −36.6 (−71.8 to −1.3)  .04  Postoperative Day  Forelimb Grip Strength, g, Mean (SD)  Differences Between Groups  Sham and SCI  Sham and NMES  SCI and NMES  Sham Group  SCI 
Group  NMES Group  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Baseline  115.4 (25.2)  110.5 (12.5)  106.0 (15.5)  4.9 (−20.8 to 30.7)  < .001  9.4 (−16.4 to 35.2)  1.0  4.5 (−22.2 to 31.1)  1.0  2  89.1 (5.4)  3.1 (5.4)  4.14 (5.39)  86.0 (77.3 to 94.6)  < .001  85.0 (76.3 to 93.6)  < .001  −1.0 (−9.9 to 7.9)  1.0  7  121.9 (22.9)  36.9 (13.5)  48.4 (24.7)  85.0 (56.4 to 113.6)  < .001  73.5 (44.8 to 102.1)  < .001  −11.6 (−41.1 to 18.0)  .95  14  127.0 (30.2)  49.4 (7.7)  52.7 (20.7)  77.6 (47.5 to 107.8)  < .001  74.2 (44.1 to 104.4)  < .001  −3.4 (−34.5 to 27.8)  1.0  21  120.3 (14.6)  47.5 (13.4)  87.3 (26.2)  72.8 (47.3 to 98.3)  < .001  32.9 (7.4 to 58.5)  .009  −39.8 (−66.2 to −13.5)  .002  28  141.3 (33.2)  66.3 (15.9)  110.3 (26.6)  75.0 (38.8 to 111.2)  < .001  31.1 (−5.0 to 67.2)  .18  −43.9 (−81.3 to −6.5)  .018  35  149.5 (23.1)  74.6 (32.5)  111.1 (17.8)  74.9 (40.8 to 109.1)  < .001  38.4 (4.3 to 72.5)  .024  −36.6 (−71.8 to −1.3)  .04  aSham group = control group without spinal cord injury, SCI group = spinal cord injury only, NMES group = spinal cord injury and neuromuscular electrical stimulation protocol. View Large At baseline, the animals showed similar values for grip test strength (total group mean: 110.8 ± 18.5 g; P = 1.00). The sham group animals had a reduction in the grip test values on postoperative day 2 (89.2 ± 5.4 g) compared to their baseline (115.4 ± 25.2 g; P = .019), returning to values similar to the baseline assessment in the remaining period of evaluations. The animals in the SCI groups had reduced muscle strength on postoperative day 2 when compared to their baseline values (P < .001) and when compared to the sham group at the same period (P < .001). The muscle strength recovery in animals from these groups occurred gradually over time. However, in the NMES group, the muscle strength recovery was more expressive from postoperative day 21 and remained until the last day of treatment, reaching higher values when compared to the SCI group. Forelimb function recovery The forelimb function recovery was assessed using the IBB Forelimb Recovery Scale at baseline and once a week during the 5-week NMES protocol. The ANOVA for repeated measures demonstrated significant main effects for groups (F = 113.2; df = 2,18; P < .001; power = 1.00), for time (F = 141.1; df = 6,108; P < .001; power = 1.00), and for the interaction between time and group (F = 40.432; df = 12,108; P < .001; power = 1.00). The results are shown in Figure 2B. At baseline, all animals showed maximum scores for forelimb function on the IBB scale (9.0 ± 0.0 points). On postoperative day 2, while the animals in the sham group remained approximate to maximum scores (8.62 ± 0.51 points), the animals that received the SCI (SCI and NMES groups) showed a significant decrease in the forelimb function scores (0.85 ± 1.5 point and 1.16 ± 1.16 point, respectively) compared to baseline values (P < .001). In both the SCI and NMES groups, forelimb function recovery occurred gradually over time, but the baseline scores were never reached, and there was no difference between the groups. Furthermore, the SCI animals showed lower scores when compared with the sham group during the 5 weeks of the protocol (P < .05). Cyclical NMES Promotes Biceps Muscle Trophism Recovery/Preservation After 
Incomplete SCI The biceps muscle trophism was measured at the end of cyclical NMES protocol using the muscle weight/body weight ratio. The ANOVA demonstrated significant main effects (F = 8.4; df = 2,21; P < .002; power = 0.93). The results are shown in Figure 3. At the end of the 5-week protocol, the animals that received treatment with cyclical NMES had higher muscle trophism values when compared to the SCI group (0.061 ± 0.005 g and 0.051 ± 0.006 g, respectively; P = .005). Furthermore, the NMES group showed no difference in muscle trophism values when compared to the sham group (0.06 ± 0.005 g and 0.06 ± 0.005 g, respectively; P = .99). It is noteworthy to say that there is no difference between the body weights of the animals in the experimental groups (SCI and NMES; P > .05). This result certifies that variation in muscle weight/body weight is related not to body weight variation but to muscle trophism. Figure 3. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on biceps muscle trophism 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for biceps muscle trophism. # = significant difference (P < .05) compared with the SCI group. Figure 3. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on biceps muscle trophism 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for biceps muscle trophism. # = significant difference (P < .05) compared with the SCI group. NMES and Muscle Trophism Cellular Signaling After Incomplete SCI Cyclical NMES increases phosphorylated Akt and p70S6K levels The Akt and p70S6K proteins are actively involved in signaling muscle hypertrophy when they are phosphorylated in the Akt/mTOR pathway. The data analysis of the phospho-Akt and phospho-p70S6K levels demonstrated significant ANOVA main effects (F = 3.5; df = 2,16; P < .05; power = 0.56; and F = 5.4; df = 2,15; P = .02; power = 0.75; respectively). Cyclical NMES treatment after SCI increased the levels of both the phospho-Akt and phospho-p70S6K proteins, as shown in Figure 4. On postoperative day 36, 24 hours after the last cyclical NMES treatment session, the animals in the NMES group had higher values for phospho-Akt levels when compared to the SCI group (495.16 ± 170.01 pg/mg of protein and 190.86 ± 64.22 pg/mg of protein, respectively; P = .047; Fig. 4A). Figure 4. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on the expression of phosphorylated serine/threonine/kinase (phospho-Akt) (A), phosphorylated ribosomal protein S6 kinase (phospho-p70S6K) (B), and phosphorylated glycogen synthase kinase-3β (phospho-GSK-3ß) (C) proteins in the biceps muscle 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for the expression of biceps muscle proteins. # = significant difference (P < .05) compared with the SCI group; * = significant difference (P < .05) compared with the sham group. Figure 4. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on the expression of phosphorylated serine/threonine/kinase (phospho-Akt) (A), phosphorylated ribosomal protein S6 kinase (phospho-p70S6K) (B), and phosphorylated glycogen synthase kinase-3β (phospho-GSK-3ß) (C) proteins in the biceps muscle 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for the expression of biceps muscle proteins. # = significant difference (P < .05) compared with the SCI group; * = significant difference (P < .05) compared with the sham group. In the levels of the phospho-p70S6K protein, a similar result was observed; the group that received NMES treatment had higher values for phospho-p70S6k levels when compared to the SCI group (1806.60 ± 833.48 pg/mg of protein and 841.19 ± 329.85 pg/mg of protein, respectively; P = .029; Fig. 4B). Cyclical NMES induces phosphorylation of GSK-3ß The GSK-3ß protein has a hypotrophic function by inhibiting the muscle hypertrophy pathway, and when phosphorylated, that function is suppressed and allows the development of the full hypertrophic Akt/mTOR pathway. The ANOVA demonstrated significant main effects (F = 5.2; df = 2,16; P = .02; power = 0.74). The 5-week NMES protocol increased the levels of phosphorylated GSK-3ß after spinal cord injury. The NMES group had higher values of phosphorylated protein when compared to the SCI group (2846.73 ± 1267.67 pg/mg of protein and 1203.49 ± 498.05 pg/mg of protein, respectively; P = .018; Fig. 4C). Discussion Despite the widespread application of NMES therapy in clinical practice, there is no consensus regarding its effectiveness in terms of improving strength and muscular trophism of partially paralyzed muscles after SCI.5,27 In addition, the biological mechanisms that underlie the effects of NMES in this condition remain unknown. In the present study, we have demonstrated that transcutaneous application of early NMES therapy increase muscle strength measured by the grip test and stimulated the biceps muscle hypertrophy measured using the muscle weight/body weight ratio, after incomplete cervical SCI in a rat model. Another interesting finding is that 5-week NMES therapy in partially paralyzed biceps muscle of the SCI-operated rats increased phospho-Akt, phospho-p70S6K, and phospho-GSK-3β levels, which participate in the muscle protein synthesis pathway, and may be linked to the gain of muscle mass and strength by the activation of the Akt/mTOR pathway. NMES Accelerates Strength Recovery and Promotes Biceps Muscle Trophism Recovery After Incomplete SCI The early NMES therapy applied after SCI in partially paralyzed muscles for 5 weeks was effective for improving muscle strength and trophism, but was not effective at improving forelimb functional recovery. The forelimb function measured using the IBB scale was not superior in NMES group, despite this, muscle strength and muscle trophism measured using the grip test and the muscle weight/body weight ratio were significantly higher in the NMES compared to the SCI group at the end of 5-week NMES protocol. In our study, the functional recovery of forelimb assessed using the IBB scale did not show greater improvement in animals that received cyclical NMES, but both the SCI and NMES groups showed spontaneous recovery, although they failed to reach the SHAM group values at the end of the 5-week protocol. Other published studies found positive results in functional recovery of forelimb or hind limb, but they involved the performance of voluntary activities, such as gait training with weight support, and functional NMES.28–32 This differences regarding functional recovery may be due to different methods of application, for example, functional NMES applied as a cointervention with functional activity or cyclical NMES applied only with the muscle contraction. Cyclical NMES stimuli alone may not be sufficient to stimulate motor learning and develop a skill, so promoting increased limb function.12,13,30 Furthermore, although the 5-week protocol was effective in improving muscle strength, a longer period of NMES may be required to ensure the increase in muscle strength and trophism is transferred to forelimb function. To our knowledge, there is only 1 other study with an animal model of SCI that addressed the effects of applying NMES to improve muscle trophism and strength conducted by Butezloff et al13 and there are few clinical studies conducted in humans with SCI. Despite the scarcity of experimental and clinical studies, the use of NMES therapy in order to improve muscle strength has some positive indications, providing beneficial effects on partially paralyzed muscle after SCI. In a systematic review carried out by de Freitas et al were found only 5 relevant clinical studies, and there is no consensus about the efficacy or not of the NMES therapy applied in this SCI population.5 In this systematic review, only 2 studies found positive results about the use of NMES therapy associated with a functional activity. Needham-Shropshire et al found an improvement in muscle strength measured by the manual muscle test in the group that received functional NMES applied in triceps brachii in association with arm cycle-ergometer.33 Harvey et al also found an increase of voluntary quadriceps strength, with 8 weeks of functional NMES therapy in association with progressive resistance training.34 We have performed a short NMES therapy protocol (5 weeks), similar to that of Butezloff et al, and even with a short period of stimulation, we had positive results which corroborated with experimental and clinical data.13,33,34 We found an increase in muscle trophism of 20% in the NMES group when compared with the SCI group, and an increase in muscle strength of 49% in the NMES group when compared with the SCI group using cyclical NMES, demonstrating that even when it is not associated with a function or activity, the NMES is able to promote the gain of muscle strength and induce muscular trophism in partially paralyzed muscles after incomplete SCI. NMES Increases Muscle Protein Synthesis Signaling After Incomplete SCI We measured the amount of phosphorylated Akt and other muscle hypertrophy pathway downstream targets in rat muscle following SCI and 5-week NMES protocol therapy. In this study, NMES therapy in partially paralyzed muscles after SCI was able to increase the levels of phosphorylated proteins Akt, p70S6K, and GSK-3β, acting in the signaling pathway of muscle protein synthesis and hypertrophy. It is well established in the literature that voluntary muscle contraction and resistance exercises can promote protein synthesis and hypertrophy mainly through the Akt/mTOR pathway.14,15,17,18 Previous study showed an activation of Akt/mTOR signaling pathway, leading to protein synthesis after 8 weeks of resistance training in humans, signaling by an increase of 140% in phosphorylated Akt.14 In addition, after repeated muscle contractile activity or a resistance training protocol, there were an increase in p70S6k phosphorylation starting 3 hours after, lasting up to 48 hours after the end of the activity and an increase of 42% in phosphorylated GSK-3β.18 Furthermore, muscle hypertrophy is associated with increased expression of insulin-like growth factor-1.17 It is a trigger for activation of phosphatidylinositol 3-kinase, which creates a lipid-binding site on the cell membrane for the Akt.14 Afterward, the Akt protein is translocated to the lipid-binding site, undergoes phosphorylation and then induces mTOR phosphorylation. Downstream mTOR signaling, occurs with the phosphorylation of 2 known regulators of protein synthesis, phospho ribosomal protein S6 kinase (p70S6K) and polyhydroxyalkanoates-1, promoting increased protein translation, while Akt also promotes phosphorylation of the glycogen synthase kinase-3β (GSK-3β), which induces protein translation by releasing its inhibition over the translation initiation of eukaryotic initiation factor 2B, thus allowing muscle growth and hypertrophy.14,15,18 In our model of incomplete SCI in rats, we found similar results with the application of NMES therapy, performing involuntary muscle contraction, with increases of 160% in phosphorylated Akt and 114% in phosphorylated p70S6K when comparing the NMES to the SCI group, thus increasing anabolism and signaling protein synthesis. Also, we found an increase of 136% 
in phosphorylation of GSK-3β when comparing the NMES to the SCI 
group. Study Strength and Limitations To the best of our knowledge, this is the first evidence showing that NMES activates the Akt pathway in partially paralyzed muscle in an animal model of incomplete SCI and promotes muscle trophism. However, there were some limitations to the present study that should be considered. An important observation is the fact that it is necessary to keep the animals under anesthesia to carry out the treatment. Despite this, it is necessary this method of application for avoid stress to the animal, and allows the application of the electrode at the correct point. Another limitation of this investigation was the lack of masking, in both the evaluation investigator and treatment therapist, although standardized testing methods were used. Furthermore, serial analysis of muscle hypertrophy protein markers at different times after SCI and adding different doses and parameters of NMES may provide additional relevant information about the effects of the NMES therapy for clinical practice and should be performed in future studies. Conclusion Electrical stimulation may be a potentially beneficial therapeutic modality to prevent muscle atrophy after SCI. In our rat model of incomplete SCI, it was demonstrated that involuntary muscle contraction induced by cyclical NMES improves muscle trophism and strength. We also found that muscle trophism may be occurred as result of increased phosphorylated protein Akt, p70S6K and GSK-3β induced by cyclical NMES therapy. With these results, we may hypothesize that involuntary contraction induced by NMES acts in a similar way to voluntary contraction with hypertrophy Akt/mTOR pathway. Additionally, this work provides some preclinical evidence about the beneficial effects of the NMES for muscle strength and trophism in partially paralyzed muscle after incomplete SCI that supports its clinical application. Author Contributions Concept/idea/research design: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, A.R.S. dos Santos, J. Ilha Writing: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Data collection: F. Bobinski, A.R.S. dos Santos, N.A.M.M. Machado-Pereira Data analysis: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Project management: G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Providing facilities/equipment: A.R.S. dos Santos Consultation (including review of manuscript before submitting): F. Bobinski Ethics Approval All procedures and surgeries followed the Guide for the Care and Use of Laboratory Animals (National Research Council of Brazil) and the guidelines of the Animal Care and Use Committee of Universidade Federal de Santa Catarina. Funding This study was funded by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (Foundation for Research and Innovation Support of the State of Santa Catarina) (FAPESC) (TO 2014TR2256). Disclosure The authors completed the ICJME Form for Disclosure of Potential Conflicts of Interest. C.C. do Espírito Santo, N.A.M.M. Machado-Pereira, and J. Ilha reported receiving grants from Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). A.R.S. dos Santos reported receiving institutional grants from CNPq, CAPES, and FAPESC and provision of writing assistance, medicines, equipment, or administrative support from Universidade Federal de Santa Catarina (UFSC). 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J Vis Exp . 2010;( 46): 2246. 24 Irvine KA, Ferguson AR, Mitchell KD et al.   The Irvine, Beatties, and Bresnahan (IBB) Forelimb Recovery Scale: an assessment of reliability and validity. Front Neurol . 2014; 5: 116. Google Scholar CrossRef Search ADS PubMed  25 Ilha J, da Cunha NB, Jaeger M et al.   Treadmill step training-induced adaptive muscular plasticity in a chronic paraplegia model. Neurosci Lett . 2011; 492: 170– 174. Google Scholar CrossRef Search ADS PubMed  26 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem . 1976; 72: 248– 254. Google Scholar CrossRef Search ADS PubMed  27 Harvey LA, Glinsky JV, Bowden JL. The effectiveness of 22 commonly administered physiotherapy interventions for people with spinal cord injury: a systematic review. Spinal Cord . 2016; 54: 914– 923. Google Scholar CrossRef Search ADS PubMed  28 Morawietz C, Moffat F. 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Early Cyclical Neuromuscular Electrical Stimulation Improves Strength and Trophism by Akt Pathway Signaling in Partially Paralyzed Biceps Muscle After Spinal Cord Injury in Rats

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American Physical Therapy Association
Copyright
© 2017 American Physical Therapy Association
ISSN
0031-9023
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1538-6724
D.O.I.
10.1093/ptj/pzx116
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Abstract

Abstract Background Electrical stimulation is often used to treat weakness in people with spinal cord injury (SCI); however its efficacy for increasing strength and trophism is weak, and the mechanisms underlying the therapeutic benefits are unknown. Objective The purpose of this study was to analyze the effects of neuromuscular electrical stimulation (NMES) on muscle function, trophism, and the Akt pathway signaling involved in muscular plasticity after incomplete SCI in rats. Design This was an experimental study. Methods Twenty-one adult female Wistar rats were divided into sham, SCI, and SCI plus NMES groups. In injured animals, SCI hemisection was induced by a surgical procedure at the C5-C7 level. The 5-week NMES protocol consisted of biceps brachii muscle stimulation 5 times per week, initiated 48 h after injury. Forepaw function and strength, biceps muscle trophism, and the expression of phosphorylated Akt, p70S6K, and GSK-3ß cellular anabolic pathway markers in stimulated muscle tissue were assessed. Results There was an increase in bicep muscle strength in the NMES group compared with the untreated SCI group, from postoperative day 21 until the end of the evaluation period. Also, there was an increase in muscle trophism in the NMES group compared with the SCI group. Forelimb function gradually recovered in both the SCI group and the NMES group, with no differences between them. Regarding muscle protein expression, the NMES group had higher values for phospho-Akt, phospho-p70S6K, and phospho-GSK-3ß than did the SCI group. Limitations The experimental findings were limited to an animal model of incomplete SCI and may not be fully generalizable to humans. Conclusions Early cyclical NMES therapy was shown to increase muscle strength and induce hypertrophy after incomplete SCI in a rat model, probably by increasing phospho-Akt, phospho-p70S6K, and phospho-GSK-3ß signaling protein synthesis. In individuals with spinal cord injury (SCI), muscle tissue is exposed to unfavorable situations, which culminate in a rapid loss of muscle mass, causing an hypotrophic state, mainly due to muscle disuse.1–3 To treat weakness in partially paralyzed muscles after a SCI, physical therapists frequently use electrical stimulation therapy.4 Despite the routine use of this mode of therapy, the efficacy of neuromuscular electrical stimulation (NMES) in increasing muscle strength and trophism is poorly studied and the mechanisms underlying the therapeutic benefits of NMES are unknown.4,5 Against the scarcity of the studies to prove the efficacy of NMES to improve strength and trophism in partially paralyzed muscle, animal models of incomplete SCI may provide some preclinical behavioral and biological evidence about the beneficial effects of the NMES in this condition. This can supports its clinical application and put forward new clinical studies with a better methodological design. However, while numerous studies have analyzed the motor and functional recovery of the anterior and posterior paws in animal models of SCI6–11 few studies have investigated the effects of NMES in such models.12,13 Beaumont et al performed 5 days of NMES associated with a functional activity (gait; functional NMES; 3 times/day), with electrodes implanted in the quadriceps and hamstring muscles in a model of SCI contusion at the T10 level.12 They found an improvement in locomotion recovery by day 7 post-SCI, measured by Basso-Beattie-Bresnahan scores.12 Another study, by Butezloff et al, in a model of SCI transection at the same level T10, consisted of 4 weeks of NMES not associated with a task, but only muscle contraction by electric stimulation (cyclical NMES; 3 days/week; 20 min/day).13 They found partial muscle hypotrophy prevention in the NMES group, showed by increased mechanical resistance and muscle mass, and a smaller deposit of fibrotic tissue in the muscles that received passive muscle contraction with cyclical NMES treatment.13 However, none of the studies analyzed the recovery of muscle strength or the possible signaling pathways involved in muscle trophism after NMES treatment. In fact, after a SCI, there is an unbalance between the muscle protein turnover that culminates with a severe muscle hypotrophy. A significant reduction of proteins participating in the muscle protein synthesis pathway was found, with 54% reduction in Akt protein, a 48% reduction in phosphorylated mammalian target of rapamycin (mTOR) protein and a 60% reduction in phosphorylated p70S6K protein.1 Surprisingly, this significant muscle loss observed in SCI population was not associated with the increase in the muscle proteolytic pathway.1,14 It is well established that physical exercise with voluntary contraction promotes muscle hypertrophy, with serine/threonine kinase (Akt)/mTOR activation being the main pathway.14–16 The trophic status of the musculature is regulated in response to changes in workload and activity, and an improvement in muscle mass “hypertrophy” is associated with increased protein synthesis.15,17,18 Despite the knowledge of this hypertrophic pathway induced by active exercise, it remains unknown whether the passive contraction induced by cyclical NMES or active-assisted contraction induced by functional NMES promotes activation of the same Akt/mTOR pathway. No studies have examined the effects and mechanisms of cyclical NMES on strength and muscle hypertrophy after SCI. To mimic the clinical condition, a rat model of incomplete SCI with cyclical NMES of partially paralyzed muscles is used to assess the possible beneficial effects of NMES therapy. Specifically, we hypothesize that cyclical NMES improves muscular function and trophism and that this muscular plasticity may be accomplished by cellular mechanisms similar to those involved in active exercises. Methods Spinal Cord Injury Procedures Adult female Wistar rats (180–200 g, n = 21) were randomly divided into 3 groups: control (sham, n = 8), SCI (n = 7), and SCI plus NMES (n = 6). The surgical procedures for preparing and performing C5-C7 SCI hemisection followed previously published methods.19 Briefly, rats were anesthetized via an intraperitoneal injection of a ketamine (87 mg/kg) and xylazine (12 mg/kg) cocktail. Once properly anesthetized, the animals’ necks were shaved and disinfected with 70% ethanol, and then the skin and musculature were carefully dissected to expose the cervical vertebrae. A complete laminectomy of the C5-C7 level was performed to expose the dorsal surface of the spinal cord, and the hemisection injury was inflicted using microscissors on the right side. The muscle and skin were closed in layers. The same surgical procedure without SCI hemisection was carried out in the sham animals. The rats were then placed in a clean home cage in a heat-controlled environment under careful monitoring by the surgeon. Recovered animals had access to food and water ad libitum. The animals received 2.5 mg/kg of enrofloxacin (antibiotic) subcutaneously (Baytril Bayer S.A., São Paulo, Brazil) immediately, and until postoperative day 3. The bladders of injured rats were expressed twice daily until micturition control returned. Neuromuscular Electrical Stimulation Therapy In the NMES group, the electrical stimulation therapy, adapted from Ambrosio et al,20 was conducted using the 2-channel Neurodyn portable device (Ibramed, São Paulo, Brazil) with animals under isoflurane (5%) inhalation anesthesia, beginning 48 hours postinjury. The therapy schedule followed a regimen of 10 min per session, once a day, 5 days per week, with a total therapy time of 5 weeks, as shown in Figure 1A. The anterior part of the right forepaws were shaved for coupling of electrodes on the skin in the biceps brachii muscle region shown in Figure 1B. Electrical stimulation parameters were as follows: biphasic asymmetric pulses; pulse duration of 150 μs; stimulation frequency of 50 Hz; and intensity adjusted to produce visible muscle contraction and progressed over time in order to maintain effective biceps brachii muscle contraction, perceived by visualization of the movement through all of the elbow flexion. Figure 1. View largeDownload slide Flowchart of the experimental time line (A) and image of the electrode placement (B). Figure 1. View largeDownload slide Flowchart of the experimental time line (A) and image of the electrode placement (B). Grip Test A grip strength meter (Columbus Instruments, Columbus, Ohio) was used to measure maximum forelimb grip strength. The meter consisted of a force transducer positioned between a metal bar. Rats were held around the midsection with 1 forelimb restrained and the other forepaw allowed to grasp the bar. The rat was gently pulled away from the device and the maximum force (g) of the grasping was recorded just before the rats release the bar. Rats were acclimated to the grip strength meter (10 trials) 1 week prior to the first testing session. Care was taken to ensure the grip was a voluntary grasp followed by release, rather than a spastic digit contracture. Testing sessions consisted of 3 successive trials, and the forelimb strength was measured once a week, until the end of the protocol.21 Irvine-Beattie-Bresnahan (IBB) Forelimb Recovery Scale To reduce the stress of the exposure to a new environment and to facilitate task learning, animals were trained to eat cereal in the test environment during the 2 weeks prior to surgery. On the test day, the animals were habituated to the laboratory conditions. Afterward, using a digital camera (Sanyo camera, digital 10) they were filmed eating 1 entire doughnut-shaped cereal piece (Kellogg's fruit loops). The animals were exposed to cereal positioned inside a cylinder with acrylic mirrors on the sides. Assessment began when the animal grasped the cereal piece, with the evaluator filming from a distance of 1 m to provide adequate zoom when the animal moved. The images were evaluated using Windows Media Player 10, slowed down to 50% or less. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping techniques.22,23 The data for each piece was collected on the score sheet described by Irvine et al23,24 and the analysis was made with just 1 piece of cereal by animal. Animals were assessed for the most common position (more than 50% of the time), referred to as the predominant position. Following these detailed assessments, the examiner filled in the score sheet for each animal and then gave a score from 0 to 9, where 0 represents the worst functionality and 9 represents normal behavior. This test was performed on baseline and on days 2, 7, 14, 21, 28, and 35 after surgery. The IBB test has strong interrater reliability (experienced raters: 0.16 ± 0.15) and validity (F = 120.89; df = 3,24; P < .00001; η2 = 0.94) to measure forelimb and fine digital control in rats, as shown by Irvine et al.24 Furthermore, the IBB test proved to be highly sensitive to the injury × time interaction (F = 7.20; df = 9,72; P <. 00001; η2 = 0.47).24 Muscle Trophism Assessment Rats were anesthetized via an intraperitoneal injection of a ketamine (87 mg/kg) and xylazine (12 mg/kg) cocktail 24 hours after the last NMES therapy session, weighed, and euthanized. The right biceps brachii muscle was dissected and weighed on a precision scale to calculate the muscle trophism, measured by the ratio of muscle weight/body weight.25 Afterward, samples of the central portion of each muscle were excised under ice, frozen in liquid nitrogen, and stored at −70°C until processed for biochemical analysis. Biochemical Analysis Muscle trophism pathway proteins were measured by an enzyme-linked immunosorbent assay (ELISA). The muscle samples were homogenized in a Ultra-Turrax Homogenizer (T-18; IKA Works, Wilmington, North Carolina) with a phosphate-buffered saline solution containing: 1 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 5 mM sodium fluoride, 6 M urea, leupeptin (10 μg/mL), pepstatin (10 μg/mL), 100 μM phenylmethylsulfonyl fluoride, aprotinin (3 μg/mL), 2.5 mM sodium pyrophosphate, and 1 mM activated sodium orthovanadate. The homogenates were transferred to 1.5-mL Eppendorf tubes and centrifuged at 3000 × g for 10 min at 4°C, and the supernatant obtained was stored at −70°C until further analysis. Total protein content was measured in the supernatant using the method of Bradford.26 The muscle tissue levels of glycogen synthase kinase-3 beta phosphorylated at S9 (phospho-GSK-3β [S9], catalog number DYC1590E), protein kinase B (Akt) phosphorylated at S473 (phospho-Akt [S473], catalog number DYC887B-2), and p70 ribosomal S6 kinase phosphorylated at T421 and S424 (phospho-p70 S6 kinase [T421/S424], catalog number DYC8965-5) were measured using sandwich ELISA according to the manufacturer's instructions (DuoSet ELISA R&D Systems, Minneapolis, Minnesota). The muscle protein levels were estimated by interpolation from a standard curve using colorimetric measurements at 450 nm (correction wavelength = 540 nm) in an ELISA plate reader (Apollo-8 LB 912; Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany). All results were expressed as pg/mg of protein. Data Analysis Comparisons among groups were performed using SPSS 20.0 software. The results of the IBB test and the grip test were analyzed using a 2-way analysis of variance (ANOVA) for repeated measures and the results of the trophism and biochemical analysis using a 1-way ANOVA. Afterward, the Tukey post hoc test for multiple comparisons was carried out. Data showed normal distribution, as analyzed by the Kolmogorov-Smirnov test. Significance levels were set at 5% (P ≤ .05). Values were expressed as the mean ± standard error of mean (SEM) or standard deviation (SD). Role of the Funding Source This study was funded by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (Foundation for Research and Innovation Support of the State of Santa Catarina) (FAPESC) (TO 2014TR2256). Financing was used to purchase ELISA kits and electrical stimulation equipment. Results NMES Accelerates Forelimb Function Recovery After Incomplete SCI Muscle strength recovery The muscle strength recovery was measured with a grip strength meter, at baseline and once a week over the 5-week cyclical NMES protocol period. The ANOVA for repeated measures demonstrated significant main effects for groups (F = 106.2; df = 2,19; P < .001; power = 1.00), for time (F = 43.1; df = 6,114; P < .001; power = 1.00) and for interaction between time and group (F = 6.2; df = 12,114; P < .001; power = 1.00) effects. The results of the groups over the period are shown in Figure 2A. In addition, the mean, standard deviation and difference between groups for injured groups are shown in the Table. Figure 2. View largeDownload slide Line plots showing the mean ± standard error of the mean for grip test muscle strength (A) and Irvine-Beattie-Bresnahan (IBB) test (B) recovery throughout the 5-week neuromuscular electrical stimulation (NMES) protocol. * = significant difference (P < .05) compared with the sham group; # = significant difference (P < .05) compared with the spinal cord injury (SCI) group. Figure 2. View largeDownload slide Line plots showing the mean ± standard error of the mean for grip test muscle strength (A) and Irvine-Beattie-Bresnahan (IBB) test (B) recovery throughout the 5-week neuromuscular electrical stimulation (NMES) protocol. * = significant difference (P < .05) compared with the sham group; # = significant difference (P < .05) compared with the spinal cord injury (SCI) group. Table. Forelimb Grip Strength (Grip Test)a Postoperative Day  Forelimb Grip Strength, g, Mean (SD)  Differences Between Groups  Sham and SCI  Sham and NMES  SCI and NMES  Sham Group  SCI 
Group  NMES Group  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Baseline  115.4 (25.2)  110.5 (12.5)  106.0 (15.5)  4.9 (−20.8 to 30.7)  < .001  9.4 (−16.4 to 35.2)  1.0  4.5 (−22.2 to 31.1)  1.0  2  89.1 (5.4)  3.1 (5.4)  4.14 (5.39)  86.0 (77.3 to 94.6)  < .001  85.0 (76.3 to 93.6)  < .001  −1.0 (−9.9 to 7.9)  1.0  7  121.9 (22.9)  36.9 (13.5)  48.4 (24.7)  85.0 (56.4 to 113.6)  < .001  73.5 (44.8 to 102.1)  < .001  −11.6 (−41.1 to 18.0)  .95  14  127.0 (30.2)  49.4 (7.7)  52.7 (20.7)  77.6 (47.5 to 107.8)  < .001  74.2 (44.1 to 104.4)  < .001  −3.4 (−34.5 to 27.8)  1.0  21  120.3 (14.6)  47.5 (13.4)  87.3 (26.2)  72.8 (47.3 to 98.3)  < .001  32.9 (7.4 to 58.5)  .009  −39.8 (−66.2 to −13.5)  .002  28  141.3 (33.2)  66.3 (15.9)  110.3 (26.6)  75.0 (38.8 to 111.2)  < .001  31.1 (−5.0 to 67.2)  .18  −43.9 (−81.3 to −6.5)  .018  35  149.5 (23.1)  74.6 (32.5)  111.1 (17.8)  74.9 (40.8 to 109.1)  < .001  38.4 (4.3 to 72.5)  .024  −36.6 (−71.8 to −1.3)  .04  Postoperative Day  Forelimb Grip Strength, g, Mean (SD)  Differences Between Groups  Sham and SCI  Sham and NMES  SCI and NMES  Sham Group  SCI 
Group  NMES Group  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Difference 
(95% CI)  P  Baseline  115.4 (25.2)  110.5 (12.5)  106.0 (15.5)  4.9 (−20.8 to 30.7)  < .001  9.4 (−16.4 to 35.2)  1.0  4.5 (−22.2 to 31.1)  1.0  2  89.1 (5.4)  3.1 (5.4)  4.14 (5.39)  86.0 (77.3 to 94.6)  < .001  85.0 (76.3 to 93.6)  < .001  −1.0 (−9.9 to 7.9)  1.0  7  121.9 (22.9)  36.9 (13.5)  48.4 (24.7)  85.0 (56.4 to 113.6)  < .001  73.5 (44.8 to 102.1)  < .001  −11.6 (−41.1 to 18.0)  .95  14  127.0 (30.2)  49.4 (7.7)  52.7 (20.7)  77.6 (47.5 to 107.8)  < .001  74.2 (44.1 to 104.4)  < .001  −3.4 (−34.5 to 27.8)  1.0  21  120.3 (14.6)  47.5 (13.4)  87.3 (26.2)  72.8 (47.3 to 98.3)  < .001  32.9 (7.4 to 58.5)  .009  −39.8 (−66.2 to −13.5)  .002  28  141.3 (33.2)  66.3 (15.9)  110.3 (26.6)  75.0 (38.8 to 111.2)  < .001  31.1 (−5.0 to 67.2)  .18  −43.9 (−81.3 to −6.5)  .018  35  149.5 (23.1)  74.6 (32.5)  111.1 (17.8)  74.9 (40.8 to 109.1)  < .001  38.4 (4.3 to 72.5)  .024  −36.6 (−71.8 to −1.3)  .04  aSham group = control group without spinal cord injury, SCI group = spinal cord injury only, NMES group = spinal cord injury and neuromuscular electrical stimulation protocol. View Large At baseline, the animals showed similar values for grip test strength (total group mean: 110.8 ± 18.5 g; P = 1.00). The sham group animals had a reduction in the grip test values on postoperative day 2 (89.2 ± 5.4 g) compared to their baseline (115.4 ± 25.2 g; P = .019), returning to values similar to the baseline assessment in the remaining period of evaluations. The animals in the SCI groups had reduced muscle strength on postoperative day 2 when compared to their baseline values (P < .001) and when compared to the sham group at the same period (P < .001). The muscle strength recovery in animals from these groups occurred gradually over time. However, in the NMES group, the muscle strength recovery was more expressive from postoperative day 21 and remained until the last day of treatment, reaching higher values when compared to the SCI group. Forelimb function recovery The forelimb function recovery was assessed using the IBB Forelimb Recovery Scale at baseline and once a week during the 5-week NMES protocol. The ANOVA for repeated measures demonstrated significant main effects for groups (F = 113.2; df = 2,18; P < .001; power = 1.00), for time (F = 141.1; df = 6,108; P < .001; power = 1.00), and for the interaction between time and group (F = 40.432; df = 12,108; P < .001; power = 1.00). The results are shown in Figure 2B. At baseline, all animals showed maximum scores for forelimb function on the IBB scale (9.0 ± 0.0 points). On postoperative day 2, while the animals in the sham group remained approximate to maximum scores (8.62 ± 0.51 points), the animals that received the SCI (SCI and NMES groups) showed a significant decrease in the forelimb function scores (0.85 ± 1.5 point and 1.16 ± 1.16 point, respectively) compared to baseline values (P < .001). In both the SCI and NMES groups, forelimb function recovery occurred gradually over time, but the baseline scores were never reached, and there was no difference between the groups. Furthermore, the SCI animals showed lower scores when compared with the sham group during the 5 weeks of the protocol (P < .05). Cyclical NMES Promotes Biceps Muscle Trophism Recovery/Preservation After 
Incomplete SCI The biceps muscle trophism was measured at the end of cyclical NMES protocol using the muscle weight/body weight ratio. The ANOVA demonstrated significant main effects (F = 8.4; df = 2,21; P < .002; power = 0.93). The results are shown in Figure 3. At the end of the 5-week protocol, the animals that received treatment with cyclical NMES had higher muscle trophism values when compared to the SCI group (0.061 ± 0.005 g and 0.051 ± 0.006 g, respectively; P = .005). Furthermore, the NMES group showed no difference in muscle trophism values when compared to the sham group (0.06 ± 0.005 g and 0.06 ± 0.005 g, respectively; P = .99). It is noteworthy to say that there is no difference between the body weights of the animals in the experimental groups (SCI and NMES; P > .05). This result certifies that variation in muscle weight/body weight is related not to body weight variation but to muscle trophism. Figure 3. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on biceps muscle trophism 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for biceps muscle trophism. # = significant difference (P < .05) compared with the SCI group. Figure 3. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on biceps muscle trophism 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for biceps muscle trophism. # = significant difference (P < .05) compared with the SCI group. NMES and Muscle Trophism Cellular Signaling After Incomplete SCI Cyclical NMES increases phosphorylated Akt and p70S6K levels The Akt and p70S6K proteins are actively involved in signaling muscle hypertrophy when they are phosphorylated in the Akt/mTOR pathway. The data analysis of the phospho-Akt and phospho-p70S6K levels demonstrated significant ANOVA main effects (F = 3.5; df = 2,16; P < .05; power = 0.56; and F = 5.4; df = 2,15; P = .02; power = 0.75; respectively). Cyclical NMES treatment after SCI increased the levels of both the phospho-Akt and phospho-p70S6K proteins, as shown in Figure 4. On postoperative day 36, 24 hours after the last cyclical NMES treatment session, the animals in the NMES group had higher values for phospho-Akt levels when compared to the SCI group (495.16 ± 170.01 pg/mg of protein and 190.86 ± 64.22 pg/mg of protein, respectively; P = .047; Fig. 4A). Figure 4. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on the expression of phosphorylated serine/threonine/kinase (phospho-Akt) (A), phosphorylated ribosomal protein S6 kinase (phospho-p70S6K) (B), and phosphorylated glycogen synthase kinase-3β (phospho-GSK-3ß) (C) proteins in the biceps muscle 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for the expression of biceps muscle proteins. # = significant difference (P < .05) compared with the SCI group; * = significant difference (P < .05) compared with the sham group. Figure 4. View largeDownload slide Effects of the 5-week neuromuscular electrical stimulation (NMES) protocol on the expression of phosphorylated serine/threonine/kinase (phospho-Akt) (A), phosphorylated ribosomal protein S6 kinase (phospho-p70S6K) (B), and phosphorylated glycogen synthase kinase-3β (phospho-GSK-3ß) (C) proteins in the biceps muscle 36 days after spinal cord injury (SCI). Bar plots show the mean ± standard error of the mean for the expression of biceps muscle proteins. # = significant difference (P < .05) compared with the SCI group; * = significant difference (P < .05) compared with the sham group. In the levels of the phospho-p70S6K protein, a similar result was observed; the group that received NMES treatment had higher values for phospho-p70S6k levels when compared to the SCI group (1806.60 ± 833.48 pg/mg of protein and 841.19 ± 329.85 pg/mg of protein, respectively; P = .029; Fig. 4B). Cyclical NMES induces phosphorylation of GSK-3ß The GSK-3ß protein has a hypotrophic function by inhibiting the muscle hypertrophy pathway, and when phosphorylated, that function is suppressed and allows the development of the full hypertrophic Akt/mTOR pathway. The ANOVA demonstrated significant main effects (F = 5.2; df = 2,16; P = .02; power = 0.74). The 5-week NMES protocol increased the levels of phosphorylated GSK-3ß after spinal cord injury. The NMES group had higher values of phosphorylated protein when compared to the SCI group (2846.73 ± 1267.67 pg/mg of protein and 1203.49 ± 498.05 pg/mg of protein, respectively; P = .018; Fig. 4C). Discussion Despite the widespread application of NMES therapy in clinical practice, there is no consensus regarding its effectiveness in terms of improving strength and muscular trophism of partially paralyzed muscles after SCI.5,27 In addition, the biological mechanisms that underlie the effects of NMES in this condition remain unknown. In the present study, we have demonstrated that transcutaneous application of early NMES therapy increase muscle strength measured by the grip test and stimulated the biceps muscle hypertrophy measured using the muscle weight/body weight ratio, after incomplete cervical SCI in a rat model. Another interesting finding is that 5-week NMES therapy in partially paralyzed biceps muscle of the SCI-operated rats increased phospho-Akt, phospho-p70S6K, and phospho-GSK-3β levels, which participate in the muscle protein synthesis pathway, and may be linked to the gain of muscle mass and strength by the activation of the Akt/mTOR pathway. NMES Accelerates Strength Recovery and Promotes Biceps Muscle Trophism Recovery After Incomplete SCI The early NMES therapy applied after SCI in partially paralyzed muscles for 5 weeks was effective for improving muscle strength and trophism, but was not effective at improving forelimb functional recovery. The forelimb function measured using the IBB scale was not superior in NMES group, despite this, muscle strength and muscle trophism measured using the grip test and the muscle weight/body weight ratio were significantly higher in the NMES compared to the SCI group at the end of 5-week NMES protocol. In our study, the functional recovery of forelimb assessed using the IBB scale did not show greater improvement in animals that received cyclical NMES, but both the SCI and NMES groups showed spontaneous recovery, although they failed to reach the SHAM group values at the end of the 5-week protocol. Other published studies found positive results in functional recovery of forelimb or hind limb, but they involved the performance of voluntary activities, such as gait training with weight support, and functional NMES.28–32 This differences regarding functional recovery may be due to different methods of application, for example, functional NMES applied as a cointervention with functional activity or cyclical NMES applied only with the muscle contraction. Cyclical NMES stimuli alone may not be sufficient to stimulate motor learning and develop a skill, so promoting increased limb function.12,13,30 Furthermore, although the 5-week protocol was effective in improving muscle strength, a longer period of NMES may be required to ensure the increase in muscle strength and trophism is transferred to forelimb function. To our knowledge, there is only 1 other study with an animal model of SCI that addressed the effects of applying NMES to improve muscle trophism and strength conducted by Butezloff et al13 and there are few clinical studies conducted in humans with SCI. Despite the scarcity of experimental and clinical studies, the use of NMES therapy in order to improve muscle strength has some positive indications, providing beneficial effects on partially paralyzed muscle after SCI. In a systematic review carried out by de Freitas et al were found only 5 relevant clinical studies, and there is no consensus about the efficacy or not of the NMES therapy applied in this SCI population.5 In this systematic review, only 2 studies found positive results about the use of NMES therapy associated with a functional activity. Needham-Shropshire et al found an improvement in muscle strength measured by the manual muscle test in the group that received functional NMES applied in triceps brachii in association with arm cycle-ergometer.33 Harvey et al also found an increase of voluntary quadriceps strength, with 8 weeks of functional NMES therapy in association with progressive resistance training.34 We have performed a short NMES therapy protocol (5 weeks), similar to that of Butezloff et al, and even with a short period of stimulation, we had positive results which corroborated with experimental and clinical data.13,33,34 We found an increase in muscle trophism of 20% in the NMES group when compared with the SCI group, and an increase in muscle strength of 49% in the NMES group when compared with the SCI group using cyclical NMES, demonstrating that even when it is not associated with a function or activity, the NMES is able to promote the gain of muscle strength and induce muscular trophism in partially paralyzed muscles after incomplete SCI. NMES Increases Muscle Protein Synthesis Signaling After Incomplete SCI We measured the amount of phosphorylated Akt and other muscle hypertrophy pathway downstream targets in rat muscle following SCI and 5-week NMES protocol therapy. In this study, NMES therapy in partially paralyzed muscles after SCI was able to increase the levels of phosphorylated proteins Akt, p70S6K, and GSK-3β, acting in the signaling pathway of muscle protein synthesis and hypertrophy. It is well established in the literature that voluntary muscle contraction and resistance exercises can promote protein synthesis and hypertrophy mainly through the Akt/mTOR pathway.14,15,17,18 Previous study showed an activation of Akt/mTOR signaling pathway, leading to protein synthesis after 8 weeks of resistance training in humans, signaling by an increase of 140% in phosphorylated Akt.14 In addition, after repeated muscle contractile activity or a resistance training protocol, there were an increase in p70S6k phosphorylation starting 3 hours after, lasting up to 48 hours after the end of the activity and an increase of 42% in phosphorylated GSK-3β.18 Furthermore, muscle hypertrophy is associated with increased expression of insulin-like growth factor-1.17 It is a trigger for activation of phosphatidylinositol 3-kinase, which creates a lipid-binding site on the cell membrane for the Akt.14 Afterward, the Akt protein is translocated to the lipid-binding site, undergoes phosphorylation and then induces mTOR phosphorylation. Downstream mTOR signaling, occurs with the phosphorylation of 2 known regulators of protein synthesis, phospho ribosomal protein S6 kinase (p70S6K) and polyhydroxyalkanoates-1, promoting increased protein translation, while Akt also promotes phosphorylation of the glycogen synthase kinase-3β (GSK-3β), which induces protein translation by releasing its inhibition over the translation initiation of eukaryotic initiation factor 2B, thus allowing muscle growth and hypertrophy.14,15,18 In our model of incomplete SCI in rats, we found similar results with the application of NMES therapy, performing involuntary muscle contraction, with increases of 160% in phosphorylated Akt and 114% in phosphorylated p70S6K when comparing the NMES to the SCI group, thus increasing anabolism and signaling protein synthesis. Also, we found an increase of 136% 
in phosphorylation of GSK-3β when comparing the NMES to the SCI 
group. Study Strength and Limitations To the best of our knowledge, this is the first evidence showing that NMES activates the Akt pathway in partially paralyzed muscle in an animal model of incomplete SCI and promotes muscle trophism. However, there were some limitations to the present study that should be considered. An important observation is the fact that it is necessary to keep the animals under anesthesia to carry out the treatment. Despite this, it is necessary this method of application for avoid stress to the animal, and allows the application of the electrode at the correct point. Another limitation of this investigation was the lack of masking, in both the evaluation investigator and treatment therapist, although standardized testing methods were used. Furthermore, serial analysis of muscle hypertrophy protein markers at different times after SCI and adding different doses and parameters of NMES may provide additional relevant information about the effects of the NMES therapy for clinical practice and should be performed in future studies. Conclusion Electrical stimulation may be a potentially beneficial therapeutic modality to prevent muscle atrophy after SCI. In our rat model of incomplete SCI, it was demonstrated that involuntary muscle contraction induced by cyclical NMES improves muscle trophism and strength. We also found that muscle trophism may be occurred as result of increased phosphorylated protein Akt, p70S6K and GSK-3β induced by cyclical NMES therapy. With these results, we may hypothesize that involuntary contraction induced by NMES acts in a similar way to voluntary contraction with hypertrophy Akt/mTOR pathway. Additionally, this work provides some preclinical evidence about the beneficial effects of the NMES for muscle strength and trophism in partially paralyzed muscle after incomplete SCI that supports its clinical application. Author Contributions Concept/idea/research design: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, A.R.S. dos Santos, J. Ilha Writing: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Data collection: F. Bobinski, A.R.S. dos Santos, N.A.M.M. Machado-Pereira Data analysis: F. Bobinski, G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Project management: G.R. de Freitas, C.C. do Espírito Santo, J. Ilha Providing facilities/equipment: A.R.S. dos Santos Consultation (including review of manuscript before submitting): F. Bobinski Ethics Approval All procedures and surgeries followed the Guide for the Care and Use of Laboratory Animals (National Research Council of Brazil) and the guidelines of the Animal Care and Use Committee of Universidade Federal de Santa Catarina. Funding This study was funded by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (Foundation for Research and Innovation Support of the State of Santa Catarina) (FAPESC) (TO 2014TR2256). Disclosure The authors completed the ICJME Form for Disclosure of Potential Conflicts of Interest. C.C. do Espírito Santo, N.A.M.M. Machado-Pereira, and J. Ilha reported receiving grants from Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). A.R.S. dos Santos reported receiving institutional grants from CNPq, CAPES, and FAPESC and provision of writing assistance, medicines, equipment, or administrative support from Universidade Federal de Santa Catarina (UFSC). 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Physical TherapyOxford University Press

Published: Mar 1, 2018

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