ELECTRICAL STIMULATION INFLUENCES SATELLITE CELL DIFFERENTIATION AFTER SCIATIC NERVE CRUSH INJURY IN RATS HUAYI XING, MD,1 MOUWANG ZHOU, MD,1 PEGGY ASSINCK, BSc,2 and NAN LIU, MD1 1 Department of Rehabilitation Medicine, Peking University Third Hospital, 49 North Garden Road, Beijing 100191, PR China 2 Graduate Program in Neuroscience, University of British Columbia, Vancouver, British Columbia, Canada Accepted 16 June 2014 ABSTRACT: Introduction: Electrical stimulation is often used to prevent muscle atrophy and preserve contractile function, but its effects on the satellite cell population after nerve injury are not well understood. In this study we aimed to determine whether satellite cell differentiation is affected by electrical stimulation after nerve crush. Methods: The sciatic nerves of Sprague-Dawley (SD) rats were crushed. Half of the injured rats received daily electrical stimulation of the gastrocnemius muscle, and the others did not. Tests for detecting paired box protein 7 (Pax7), myogenic differentiation antigen (MyoD), embryonic myosin heavy chain (eMyHC), and force production were performed 2, 4, and 6 weeks after injury. Results: More Pax71/MyoD1 nuclei in stimulated muscles were observed than in non-stimulated muscles. eMyHC expression was elevated in stimulated muscles and correlated positively with enhanced force production. Conclusions: Increased satellite cell differentiation is correlated with preserved muscle function in response to electrical stimulation after nerve injury. Muscle Nerve 51: 400–411, 2015
Satellite cells are a specialized subset of mononuclear cells that associate closely with mature muscle fibers and are responsible for muscle growth and repair.1,2 Although normally quiescent, satellite cells are activated by muscle damage, which causes them to proliferate and differentiate to form fusion-competent myoblasts.3 Paired box protein 7 (Pax7), a transcription factor, marks both quiescent and activated satellite cells,4–6 whereas myogenic differentiation antigen (MyoD) marks differentiated satellite cells.7 MyoD expression is necessary for initiation of myogenesis and thus indicates irreversible differentiation of satellite cells.8,9 In adult skeletal muscles, terminally differAbbreviations: ANOVA, analysis of variance; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; eMyHC, embryonic myosin heavy chain; EITS, experimental intermediary toe spread; EPL, experimental print length; ETS, experimental toe spread; GAPDH, glyceraldehyde 3phosphate dehydrogenase; H&E, hematoxylin–eosin; HGF, hepatocyte growth factor; HPF, high-power field; MMP-2, matrix metalloproteinase-2; MyoD, myogenic differentiation antigen; NAUC, normalized area under the curve; NITS, normal intermediary toe spread; NO, nitric oxide; NOS, nitric oxide synthase; NPF, normalized peak force; NPL, normal print length; NTS, normal toe spread; OCT, optimum cutting temperature compound; OD, optical density; Pax7, paired box protein 7; PBS, phosphate-buffered saline; PCSA, physiological cross-sectional area; SD, Sprague-Dawley; SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; SFI, sciatic function index; SEM, standard error of the mean; wpi, weeks post-injury Key words: electrical stimulation; muscle atrophy; muscle transcription factor; peripheral nerve injury; skeletal muscle satellite cells This study was supported by a grant from the National Natural Science Foundation of China (81071602). Correspondence to: M. Zhou; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 20 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24322
400
Satellite Cell Differentiation
entiated satellite cells can fuse to their associated myofibers, express embryonic myosin heavy chain (eMyHC; a special type of myosin heavy chain that is expressed exclusively during embryonic development and muscle regeneration), and function in muscle contraction.10,11 For this reason, eMyHCcontaining fibers are regarded as a marker of successful regeneration induced by satellite cell differentiation.10 Satellite cells are activated in response to specific stimuli, such as local muscle damage.2,3 In contrast, in nerve injury, satellite cells in the denervated muscle do not appear to respond in the same manner and therefore cannot compensate for the loss of muscle mass that occurs during post-injury atrophy.12,13 Reparative myogenesis is limited after nerve injury. Due to the failure of satellite cells to become terminally differentiated, only abnormally small myotubes are formed.13 Furthermore, in long-term denervated skeletal muscles of adult rats, both the population and local density of satellite cells are reduced progressively due to exhaustion of the satellite cell pool,14,15 which partly explains the irreversible decline of muscle volume and function after nerve injury. Electrical stimulation is used therapeutically in nerve injury patients to prevent muscle atrophy and preserve contractile function.16 Clinical studies have shown that the degeneration of human muscle fibers that follows long-term denervation is reversed by functional electrical stimulation training17–20 and that the contractile apparatus and myofiber excitability are considerably improved.17,21 The crosssectional area of partially denervated muscle is also restored gradually in response to long-term electrical stimulation.18,20 These findings have been replicated in animal experiments, suggesting that electrical stimulation maintains muscle force and mass,22 accelerates functional recovery,23 and promotes behavioral recovery.24 The underlying mechanism responsible for these beneficial effects remains uncertain. Several studies have shown molecular and metabolic changes in stimulated muscles, such as increased expression of mitochondrial proteins25 and matrix metalloproteinase-2,26 reduced apoptosis27,28 and atrogin-1 synthesis,29 and enhanced glucose metabolism.30 However, none of these studies have addressed the possible influence of electrical stimulation on satellite cells in denervated muscles. MUSCLE & NERVE
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Electrical stimulation has been reported to improve satellite cell differentiation in both normal animals and animals with unloading-induced muscle atrophy.31–33 Satellite cell differentiation is greatly elevated by electrical stimulation in healthy rats,31,32 and this effect is more obvious in adult and aging rats.32 In another study, compared with atrophic controls, the percentage of differentiated satellite cells was increased by low-frequency electrical stimulation during hindlimb suspension; lowfrequency electrical stimulation also resulted in restoration of myofiber cross-sectional area.33 Based on those results, we hypothesized that electrical stimulation also improves satellite cell differentiation after nerve injury and that enhanced satellite cell differentiation consequently promotes fiber regeneration and functional restoration. In this study we aimed to determine: (1) whether satellite cell differentiation is activated by electrical stimulation after nerve injury; (2) whether successful myogenesis occurs in electrically stimulated muscles; and (3) whether improvement in muscle contractile function correlates with the regenerative myogenesis induced by satellite cell differentiation. We hypothesized that the percentage of differentiated satellite cells that coexpress Pax7 and MyoD would increase with electrical stimulation after nerve injury, that there would be significantly more regenerating myofibers expressing eMyHC in the stimulated muscles than in non-stimulated controls, and that this increase in eMyHC expression would correlate positively with restoration of contractile function. METHODS
Researchers blinded to the experimental groups performed all of the measurements. Seventy-two adult male Sprague-Dawley rats (Department of Laboratory Animal Science, Peking University Health Science Center, Beijing, China), weighing 291.43 6 1.46 g (mean 6 SEM), were used in this study. All animal procedures were in strict accordance with the recommendations of the Chinese Laboratory Animal Requirements of Environment and Housing Facilities. The protocols were approved by the Committee on the Ethics of Animal Experiments of Peking University. All efforts were made to minimize animal suffering. The rats were housed individually under controlled environmental conditions (22 C with alternating 12-h light and dark cycles) and received standard rat chow and water ad libitum. The animals were assigned randomly to 1 of the following 3 groups (n 5 24 each): (1) shamoperated controls (Sham); (2) sciatic nerve crush injury (Injury); or (3) sciatic nerve crush injury 1 daily electrical stimulation (Injury1Stim). Due Animals.
Satellite Cell Differentiation
to the size of the experiment, several cohorts were studied, but care was taken to ensure that equal numbers of animals from each group were studied for each cohort. All animals survived the experimental period without adverse effects and were included in the data analysis. Sciatic Nerve Crush Injury Procedure. Surgery was performed as described previously.34 In brief, animals were anesthetized using intraperitoneal sodium pentobarbital (5 mg per 100 g body weight). Rats in the Injury and Injury1Stim groups were shaved, and their skin was prepared with 75% alcohol; then, the left sciatic nerve was exposed and crushed 10 mm above the bifurcation for 30 seconds with a serrated clamp that exerted a force of 100 N. The incision was then closed with 4-0 nylon sutures. The same procedures were performed for rats in the Sham group, except that the nerves were not crushed. Aseptic techniques were used to avoid infection, and the animals’ temperature was maintained at 37 C throughout the procedures. Calculation of Sciatic Function Index. The sciatic function index (SFI) was calculated 3, 7, 14, 28, and 42 days after nerve crush as described by GigoBenato et al.16 The hind paws of the rats were smeared with black ink. The animals were then allowed to walk down a confined corridor (42 cm long, 8.2 cm wide) with a dark shelter at the end. A piece of white paper was placed on the floor of the corridor to capture the hind footprints. For each pair of footprints, the following 3 measurements were collected for the experimental (E) and normal (N) sides (Fig. 1): (1) print length (EPL/NPL, distance from the heel to the third toe); (2) toe spread (ETS/NTS, distance from the first to the fifth toe); and (3) intermediary toe spread (EITS/NITS, distance from the second to the fourth toe). The SFI was calculated according to the following equation: EPL2NPL ETS2NTS 1109:5 SFI5238:3 NPL NTS EITS2NITS 28:8 113:3 NITS
Electrical Stimulation. In the Injury1Stim group, electrical stimulation began 1 day post-injury and was applied daily thereafter until the experiment ended. After the rats were anesthetized lightly using intraperitoneal sodium pentobarbital (3 mg per 100 g body weight), 2 surface electrodes were fixed to the skin of the left leg over the gastrocnemius muscle. The electrodes were connected to a stimulator (ZS Dichuang, Beijing, China) set to generate square-wave pulses for 30 min (parameters: pulse rate 2 HZ; pulse width 300 ms; voltage MUSCLE & NERVE
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FIGURE 1. Typical footprints of a rat after left sciatic nerve crush, showing the three measurements for each footprint. L, left; R, right; EPL/NPL, experimental/normal print length; ETS/ NTS, experimental/normal toe spread; EITS/NITS, experimental/normal intermediary toe spread.
25 V; current 1 mA) in accordance with previously described protocols.35,36 Animals in the Sham and Injury groups received only intraperitoneal sodium pentobarbital every day without any stimulation. Harvesting of Muscle. Animals were chosen randomly from each of the 3 groups and euthanized at 2, 4, and 6 weeks post-injury (wpi) (n 5 8 from each group at each time-point). At death, the left gastrocnemius muscle of each animal was excised rapidly. The resting muscle length and wet mass were measured, and the contractile properties were tested immediately by muscle force production. The muscle sample was then cut lengthwise into 3 equal parts for immunofluorescence, immunohistochemistry, and Western blot analyses. Muscle Force Production. Muscle force production was measured as described previously.3 Muscle was attached to a tissue support with stimulating electrodes and bathed in fresh Krebs–Ringer bicarbonate buffer.3 The optimal length was determined by increasing the initial tension in 0.2-g increments and applying a single electrical pulse using a stimulator (ZS Dichuang, Beijing, China) (parameters: pulse rate 1 HZ; duration 1 ms; voltage 30 V). Force production was evaluated by maximally stimulating the muscle for 10 min. The following parameters were used: train rate 2/min; train duration 10 s; pulse rate 100 HZ; duration 0.1 ms; and voltage 30 V. An isometric force transducer (ZS Dichuang, Beijing, China) was used to monitor 402
Satellite Cell Differentiation
muscle contraction. The converted digital signals were captured and analyzed using a specialized BL420E1 software system (TaiMeng, Chengdu, China). The signals were recorded at a sampling frequency of 100 HZ and were not filtered. For each muscle, the peak force (force at the highest point of the contraction curve, reflecting the maximum strength of the muscle (in grams) and the area under the curve (g s) during the 10-minute intermittent stimulation (reflecting the co-effect of the muscle’s maximum strength as well as resistance to fatigue) were recorded and then normalized to the physiological cross-sectional area (PCSA) of the muscle.37–40 PCSA (cm2) 5 muscle mass (g) / [muscle length (cm) 1.06 g/cm3], with 1.06 g/cm3 representing the average density of mammalian skeletal muscle. Normalized peak force (NPF, in N/cm2) 5 peak force (g) 0.00981 (N/g) / PCSA (cm2). Normalized area under the curve (NAUC, in N s/cm2) 5 area under the curve (g s) 0.00981 (N/g) / PCSA (cm2). Immunofluorescence for Pax7 and MyoD. Muscle was embedded in OCT (Sakura, Torrance, California) and frozen in liquid nitrogen. Cross-sections 8 lm thick were prepared for immunostaining using a microtome (Leica, Wetzlar, Germany). Three serial cross-sections taken at the midbelly of the muscle were collected from each animal and prepared for immunostaining. Muscle sections were permeabilized with phosphate-buffered saline (PBS) containing 0.3% Triton X-100, blocked with 1% bovine serum albumin (BSA)–PBS, and incubated with primary antibodies. To colocalize Pax7 and MyoD, a rabbit polyclonal antibody against Pax7 (Abcam, Cambridge, UK; 1:200) and a mouse monoclonal antibody against MyoD (Abcam; 1:200) were used on the same section. Goat anti-rabbit IgG-conjugated PE (Molecular Probes, Carlsbad, California) and goat anti-mouse IgG-conjugated DyLight 488 (Abcam) were chosen as secondary antibodies according to the host species of the primary antibodies. Nuclei were visualized using Hoechst 33342 (Sigma-Aldrich, St. Louis, Missouri) staining in the last step. Only nuclei that stained positively with both Pax7 and MyoD (Pax71/MyoD1) were identified as differentiated satellite cell nuclei. The percentage of Pax71/MyoD1 satellite cell nuclei to total nuclei was then calculated under a fluorescence microscope (Leica) based on 5 randomly chosen41 high-power fields (HPFs; 3400 primary magnification) for each muscle section. Hematoxylin–Eosin Staining and Immunohistochemistry for eMyHC. Muscle was fixed in 10% formalin for
12 h and embedded in paraffin. Sections 5 lm thick were cut with a microtome (Leica). Four serial crosssections at the midbelly of the muscle were prepared MUSCLE & NERVE
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from each animal, 1 for hematoxylin–eosin (H&E) staining and 3 for immunohistochemical detection of eMyHC. H&E staining was performed to show the extent of muscle atrophy and central nuclei (newly fused satellite cells). For immunohistochemistry, the slides were incubated in 3% methanol–H2O2 before staining. A mouse monoclonal antibody against eMyHC (Santa Cruz Biotechnology, Dallas, Texas; 1:100) was used as the primary antibody. The sections were incubated for 2 h at room temperature, then washed and reacted with a secondary antibody conjugated with horseradish peroxidase (goat anti-mouse IgG; Abcam). Diaminobenzidine tetrahydrochloride was used as a chromogen to localize the target antigen. The percentage of eMyHC-positive fibers to total fibers was calculated under a light microscope (Nikon, Tokyo, Japan) using an average of 5 randomly chosen41 HPFs for each section. Images of sections after immunofluorescence and immunohistochemical staining were captured with a microscope camera system (Leica). Five non-overlapping images were chosen randomly from each muscle section according to the following procedure.41 A picture of the whole cross-section was captured, and squares were drawn and labeled on the picture with numbers from 1 to 50. Five numbers were then selected using a random number table, and the squares corresponding to the selected numbers were analyzed. The total number of nuclei or fibers in each image was counted, after which we counted the positively stained nuclei or fibers. In each muscle sample, the total number and positively stained number of all HPFs were averaged. Calculated percentages 5 (average number / average total number) 3 100%. One investigator performed all of the counting, and all slides were coded by another investigator, thus blinding the person responsible for the counting to the slide identities. Image Analysis.
Western Blotting. The muscle was lysed in RIPA buffer (CWBIO, Beijing, China), and total tissue protein was extracted. Western blot analysis was performed as described previously.27,42 In brief, total protein concentration was determined using bicinchoninic acid assays. Aliquots dissolved in sodium dodecylsulfate buffer were analyzed using 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 30 lg/lane), followed by Western blotting. Primary antibodies used for protein detection included mouse anti-rat MyoD monoclonal antibody (Abcam; 1:500) and mouse anti-rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Abcam; 1:500, as a loading control). Goat anti-mouse IgG-conjugated IRdye 800 (LI-COR, Lincoln, Nebraska) was used as a secondary antibody. All bands were scanned densitometrically and evaluated quantitaSatellite Cell Differentiation
Table 1. Sciatic function index (%) at different time-points.
3 days 7 days 14 days 28 days 42 days
Sham
Injury
Injury1Stim
26.03 6 0.64 25.86 6 0.83 25.56 6 0.77 26.32 6 0.76 25.70 6 0.71
283.51 6 1.64* 284.53 6 1.80* 283.15 6 3.36* 284.70 6 2.77* 284.04 6 2.45*
284.70 6 2.59* 285.05 6 2.33* 285.55 6 3.17* 283.80 6 2.79* 285.00 6 2.08*
Data presented as mean 6 SEM. *P < 0.05, significantly different from Sham at the same time-point.
tively using the Odyssey system (LI-COR), such that the ratio of MyoD band density to GAPDH band density (relative optical density, OD) could be calculated using ImageJ (National Institutes of Health, Bethesda, Maryland). The average density ratios of the different groups were compared using statistical analysis. The samples from the Injury, Injury1Stim, and Sham-6 wpi groups were run simultaneously on the same gel. The samples from the Sham-2 wpi and Sham-4 wpi groups were run subsequently on another gel. Statistics. SPSS SamplePower (IBM, Armonk, New York) was used to determine the sample size of each group sufficient for statistical significance with a power of 0.90 (1 – b 5 0.9). All data were distributed normally and are presented as the mean 6 SEM. The data were analyzed using 2-way analysis of variance (ANOVA). When a significant F-ratio was found, a Bonferroni post-hoc test was used to identify the differences. Linear regression analysis was performed to evaluate the relationship between the percentage of eMyHC-positive fibers and normalized peak force / normalized area under the curve (NPF/NAUC) (separately). Differences were considered significant when P < 0.05, and analyses were performed using SPSS, version 16.0 (IBM). RESULTS SFI and Muscle Mass. SFI data are listed in Table 1. Both the Injury and Injury1Stim groups showed an approximately 80% loss of sciatic nerve function, which was significantly greater than in the Sham group. No significant difference in SFI was observed between the Injury and Injury1Stim groups at any time-point. The muscle length and mass data are listed in Table 2. Muscle mass differed between the 3 groups at each time-point, with the highest muscle mass in the Sham group, a lower muscle mass in the Injury1Stim group, and the lowest in the Injury group. Increase in Satellite Cell Differentiation in Stimulated Muscle after Nerve Crush Injury. Typical images showing differentiated (Pax71/MyoD1) satellite cells in muscle cross-sections from the 3 groups MUSCLE & NERVE
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Table 2. Length and mass of muscle samples.
2 wpi 4 wpi 6 wpi
Sham Length (mm) Mass (g)
Injury Length (mm) Mass (g)
Injury1Stim Length (mm) Mass (g)
34.24 6 1.79 2.37 6 0.14 37.05 6 1.96 2.28 6 0.18 35.14 6 1.78 2.47 6 0.09
37.79 6 1.69 1.12 6 0.15* 35.94 6 1.74 1.02 6 0.19* 35.13 6 1.92 0.96 6 0.18*
38.15 6 2.14 1.87 6 0.21*† 36.27 6 2.00 1.64 6 0.16*† 36.87 6 1.51 1.78 6 0.22*†
Data presented as mean 6 SEM. wpi, weeks post-injury. *P
Satellite cells are a specialized subset of mononuclear cells that associate closely with mature muscle fibers and are responsible for muscle growth and repair.1,2 Although normally quiescent, satellite cells are activated by muscle damage, which causes them to proliferate and differentiate to form fusion-competent myoblasts.3 Paired box protein 7 (Pax7), a transcription factor, marks both quiescent and activated satellite cells,4–6 whereas myogenic differentiation antigen (MyoD) marks differentiated satellite cells.7 MyoD expression is necessary for initiation of myogenesis and thus indicates irreversible differentiation of satellite cells.8,9 In adult skeletal muscles, terminally differAbbreviations: ANOVA, analysis of variance; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; eMyHC, embryonic myosin heavy chain; EITS, experimental intermediary toe spread; EPL, experimental print length; ETS, experimental toe spread; GAPDH, glyceraldehyde 3phosphate dehydrogenase; H&E, hematoxylin–eosin; HGF, hepatocyte growth factor; HPF, high-power field; MMP-2, matrix metalloproteinase-2; MyoD, myogenic differentiation antigen; NAUC, normalized area under the curve; NITS, normal intermediary toe spread; NO, nitric oxide; NOS, nitric oxide synthase; NPF, normalized peak force; NPL, normal print length; NTS, normal toe spread; OCT, optimum cutting temperature compound; OD, optical density; Pax7, paired box protein 7; PBS, phosphate-buffered saline; PCSA, physiological cross-sectional area; SD, Sprague-Dawley; SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; SFI, sciatic function index; SEM, standard error of the mean; wpi, weeks post-injury Key words: electrical stimulation; muscle atrophy; muscle transcription factor; peripheral nerve injury; skeletal muscle satellite cells This study was supported by a grant from the National Natural Science Foundation of China (81071602). Correspondence to: M. Zhou; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 20 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24322
400
Satellite Cell Differentiation
entiated satellite cells can fuse to their associated myofibers, express embryonic myosin heavy chain (eMyHC; a special type of myosin heavy chain that is expressed exclusively during embryonic development and muscle regeneration), and function in muscle contraction.10,11 For this reason, eMyHCcontaining fibers are regarded as a marker of successful regeneration induced by satellite cell differentiation.10 Satellite cells are activated in response to specific stimuli, such as local muscle damage.2,3 In contrast, in nerve injury, satellite cells in the denervated muscle do not appear to respond in the same manner and therefore cannot compensate for the loss of muscle mass that occurs during post-injury atrophy.12,13 Reparative myogenesis is limited after nerve injury. Due to the failure of satellite cells to become terminally differentiated, only abnormally small myotubes are formed.13 Furthermore, in long-term denervated skeletal muscles of adult rats, both the population and local density of satellite cells are reduced progressively due to exhaustion of the satellite cell pool,14,15 which partly explains the irreversible decline of muscle volume and function after nerve injury. Electrical stimulation is used therapeutically in nerve injury patients to prevent muscle atrophy and preserve contractile function.16 Clinical studies have shown that the degeneration of human muscle fibers that follows long-term denervation is reversed by functional electrical stimulation training17–20 and that the contractile apparatus and myofiber excitability are considerably improved.17,21 The crosssectional area of partially denervated muscle is also restored gradually in response to long-term electrical stimulation.18,20 These findings have been replicated in animal experiments, suggesting that electrical stimulation maintains muscle force and mass,22 accelerates functional recovery,23 and promotes behavioral recovery.24 The underlying mechanism responsible for these beneficial effects remains uncertain. Several studies have shown molecular and metabolic changes in stimulated muscles, such as increased expression of mitochondrial proteins25 and matrix metalloproteinase-2,26 reduced apoptosis27,28 and atrogin-1 synthesis,29 and enhanced glucose metabolism.30 However, none of these studies have addressed the possible influence of electrical stimulation on satellite cells in denervated muscles. MUSCLE & NERVE
March 2015
Electrical stimulation has been reported to improve satellite cell differentiation in both normal animals and animals with unloading-induced muscle atrophy.31–33 Satellite cell differentiation is greatly elevated by electrical stimulation in healthy rats,31,32 and this effect is more obvious in adult and aging rats.32 In another study, compared with atrophic controls, the percentage of differentiated satellite cells was increased by low-frequency electrical stimulation during hindlimb suspension; lowfrequency electrical stimulation also resulted in restoration of myofiber cross-sectional area.33 Based on those results, we hypothesized that electrical stimulation also improves satellite cell differentiation after nerve injury and that enhanced satellite cell differentiation consequently promotes fiber regeneration and functional restoration. In this study we aimed to determine: (1) whether satellite cell differentiation is activated by electrical stimulation after nerve injury; (2) whether successful myogenesis occurs in electrically stimulated muscles; and (3) whether improvement in muscle contractile function correlates with the regenerative myogenesis induced by satellite cell differentiation. We hypothesized that the percentage of differentiated satellite cells that coexpress Pax7 and MyoD would increase with electrical stimulation after nerve injury, that there would be significantly more regenerating myofibers expressing eMyHC in the stimulated muscles than in non-stimulated controls, and that this increase in eMyHC expression would correlate positively with restoration of contractile function. METHODS
Researchers blinded to the experimental groups performed all of the measurements. Seventy-two adult male Sprague-Dawley rats (Department of Laboratory Animal Science, Peking University Health Science Center, Beijing, China), weighing 291.43 6 1.46 g (mean 6 SEM), were used in this study. All animal procedures were in strict accordance with the recommendations of the Chinese Laboratory Animal Requirements of Environment and Housing Facilities. The protocols were approved by the Committee on the Ethics of Animal Experiments of Peking University. All efforts were made to minimize animal suffering. The rats were housed individually under controlled environmental conditions (22 C with alternating 12-h light and dark cycles) and received standard rat chow and water ad libitum. The animals were assigned randomly to 1 of the following 3 groups (n 5 24 each): (1) shamoperated controls (Sham); (2) sciatic nerve crush injury (Injury); or (3) sciatic nerve crush injury 1 daily electrical stimulation (Injury1Stim). Due Animals.
Satellite Cell Differentiation
to the size of the experiment, several cohorts were studied, but care was taken to ensure that equal numbers of animals from each group were studied for each cohort. All animals survived the experimental period without adverse effects and were included in the data analysis. Sciatic Nerve Crush Injury Procedure. Surgery was performed as described previously.34 In brief, animals were anesthetized using intraperitoneal sodium pentobarbital (5 mg per 100 g body weight). Rats in the Injury and Injury1Stim groups were shaved, and their skin was prepared with 75% alcohol; then, the left sciatic nerve was exposed and crushed 10 mm above the bifurcation for 30 seconds with a serrated clamp that exerted a force of 100 N. The incision was then closed with 4-0 nylon sutures. The same procedures were performed for rats in the Sham group, except that the nerves were not crushed. Aseptic techniques were used to avoid infection, and the animals’ temperature was maintained at 37 C throughout the procedures. Calculation of Sciatic Function Index. The sciatic function index (SFI) was calculated 3, 7, 14, 28, and 42 days after nerve crush as described by GigoBenato et al.16 The hind paws of the rats were smeared with black ink. The animals were then allowed to walk down a confined corridor (42 cm long, 8.2 cm wide) with a dark shelter at the end. A piece of white paper was placed on the floor of the corridor to capture the hind footprints. For each pair of footprints, the following 3 measurements were collected for the experimental (E) and normal (N) sides (Fig. 1): (1) print length (EPL/NPL, distance from the heel to the third toe); (2) toe spread (ETS/NTS, distance from the first to the fifth toe); and (3) intermediary toe spread (EITS/NITS, distance from the second to the fourth toe). The SFI was calculated according to the following equation: EPL2NPL ETS2NTS 1109:5 SFI5238:3 NPL NTS EITS2NITS 28:8 113:3 NITS
Electrical Stimulation. In the Injury1Stim group, electrical stimulation began 1 day post-injury and was applied daily thereafter until the experiment ended. After the rats were anesthetized lightly using intraperitoneal sodium pentobarbital (3 mg per 100 g body weight), 2 surface electrodes were fixed to the skin of the left leg over the gastrocnemius muscle. The electrodes were connected to a stimulator (ZS Dichuang, Beijing, China) set to generate square-wave pulses for 30 min (parameters: pulse rate 2 HZ; pulse width 300 ms; voltage MUSCLE & NERVE
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FIGURE 1. Typical footprints of a rat after left sciatic nerve crush, showing the three measurements for each footprint. L, left; R, right; EPL/NPL, experimental/normal print length; ETS/ NTS, experimental/normal toe spread; EITS/NITS, experimental/normal intermediary toe spread.
25 V; current 1 mA) in accordance with previously described protocols.35,36 Animals in the Sham and Injury groups received only intraperitoneal sodium pentobarbital every day without any stimulation. Harvesting of Muscle. Animals were chosen randomly from each of the 3 groups and euthanized at 2, 4, and 6 weeks post-injury (wpi) (n 5 8 from each group at each time-point). At death, the left gastrocnemius muscle of each animal was excised rapidly. The resting muscle length and wet mass were measured, and the contractile properties were tested immediately by muscle force production. The muscle sample was then cut lengthwise into 3 equal parts for immunofluorescence, immunohistochemistry, and Western blot analyses. Muscle Force Production. Muscle force production was measured as described previously.3 Muscle was attached to a tissue support with stimulating electrodes and bathed in fresh Krebs–Ringer bicarbonate buffer.3 The optimal length was determined by increasing the initial tension in 0.2-g increments and applying a single electrical pulse using a stimulator (ZS Dichuang, Beijing, China) (parameters: pulse rate 1 HZ; duration 1 ms; voltage 30 V). Force production was evaluated by maximally stimulating the muscle for 10 min. The following parameters were used: train rate 2/min; train duration 10 s; pulse rate 100 HZ; duration 0.1 ms; and voltage 30 V. An isometric force transducer (ZS Dichuang, Beijing, China) was used to monitor 402
Satellite Cell Differentiation
muscle contraction. The converted digital signals were captured and analyzed using a specialized BL420E1 software system (TaiMeng, Chengdu, China). The signals were recorded at a sampling frequency of 100 HZ and were not filtered. For each muscle, the peak force (force at the highest point of the contraction curve, reflecting the maximum strength of the muscle (in grams) and the area under the curve (g s) during the 10-minute intermittent stimulation (reflecting the co-effect of the muscle’s maximum strength as well as resistance to fatigue) were recorded and then normalized to the physiological cross-sectional area (PCSA) of the muscle.37–40 PCSA (cm2) 5 muscle mass (g) / [muscle length (cm) 1.06 g/cm3], with 1.06 g/cm3 representing the average density of mammalian skeletal muscle. Normalized peak force (NPF, in N/cm2) 5 peak force (g) 0.00981 (N/g) / PCSA (cm2). Normalized area under the curve (NAUC, in N s/cm2) 5 area under the curve (g s) 0.00981 (N/g) / PCSA (cm2). Immunofluorescence for Pax7 and MyoD. Muscle was embedded in OCT (Sakura, Torrance, California) and frozen in liquid nitrogen. Cross-sections 8 lm thick were prepared for immunostaining using a microtome (Leica, Wetzlar, Germany). Three serial cross-sections taken at the midbelly of the muscle were collected from each animal and prepared for immunostaining. Muscle sections were permeabilized with phosphate-buffered saline (PBS) containing 0.3% Triton X-100, blocked with 1% bovine serum albumin (BSA)–PBS, and incubated with primary antibodies. To colocalize Pax7 and MyoD, a rabbit polyclonal antibody against Pax7 (Abcam, Cambridge, UK; 1:200) and a mouse monoclonal antibody against MyoD (Abcam; 1:200) were used on the same section. Goat anti-rabbit IgG-conjugated PE (Molecular Probes, Carlsbad, California) and goat anti-mouse IgG-conjugated DyLight 488 (Abcam) were chosen as secondary antibodies according to the host species of the primary antibodies. Nuclei were visualized using Hoechst 33342 (Sigma-Aldrich, St. Louis, Missouri) staining in the last step. Only nuclei that stained positively with both Pax7 and MyoD (Pax71/MyoD1) were identified as differentiated satellite cell nuclei. The percentage of Pax71/MyoD1 satellite cell nuclei to total nuclei was then calculated under a fluorescence microscope (Leica) based on 5 randomly chosen41 high-power fields (HPFs; 3400 primary magnification) for each muscle section. Hematoxylin–Eosin Staining and Immunohistochemistry for eMyHC. Muscle was fixed in 10% formalin for
12 h and embedded in paraffin. Sections 5 lm thick were cut with a microtome (Leica). Four serial crosssections at the midbelly of the muscle were prepared MUSCLE & NERVE
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from each animal, 1 for hematoxylin–eosin (H&E) staining and 3 for immunohistochemical detection of eMyHC. H&E staining was performed to show the extent of muscle atrophy and central nuclei (newly fused satellite cells). For immunohistochemistry, the slides were incubated in 3% methanol–H2O2 before staining. A mouse monoclonal antibody against eMyHC (Santa Cruz Biotechnology, Dallas, Texas; 1:100) was used as the primary antibody. The sections were incubated for 2 h at room temperature, then washed and reacted with a secondary antibody conjugated with horseradish peroxidase (goat anti-mouse IgG; Abcam). Diaminobenzidine tetrahydrochloride was used as a chromogen to localize the target antigen. The percentage of eMyHC-positive fibers to total fibers was calculated under a light microscope (Nikon, Tokyo, Japan) using an average of 5 randomly chosen41 HPFs for each section. Images of sections after immunofluorescence and immunohistochemical staining were captured with a microscope camera system (Leica). Five non-overlapping images were chosen randomly from each muscle section according to the following procedure.41 A picture of the whole cross-section was captured, and squares were drawn and labeled on the picture with numbers from 1 to 50. Five numbers were then selected using a random number table, and the squares corresponding to the selected numbers were analyzed. The total number of nuclei or fibers in each image was counted, after which we counted the positively stained nuclei or fibers. In each muscle sample, the total number and positively stained number of all HPFs were averaged. Calculated percentages 5 (average number / average total number) 3 100%. One investigator performed all of the counting, and all slides were coded by another investigator, thus blinding the person responsible for the counting to the slide identities. Image Analysis.
Western Blotting. The muscle was lysed in RIPA buffer (CWBIO, Beijing, China), and total tissue protein was extracted. Western blot analysis was performed as described previously.27,42 In brief, total protein concentration was determined using bicinchoninic acid assays. Aliquots dissolved in sodium dodecylsulfate buffer were analyzed using 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 30 lg/lane), followed by Western blotting. Primary antibodies used for protein detection included mouse anti-rat MyoD monoclonal antibody (Abcam; 1:500) and mouse anti-rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Abcam; 1:500, as a loading control). Goat anti-mouse IgG-conjugated IRdye 800 (LI-COR, Lincoln, Nebraska) was used as a secondary antibody. All bands were scanned densitometrically and evaluated quantitaSatellite Cell Differentiation
Table 1. Sciatic function index (%) at different time-points.
3 days 7 days 14 days 28 days 42 days
Sham
Injury
Injury1Stim
26.03 6 0.64 25.86 6 0.83 25.56 6 0.77 26.32 6 0.76 25.70 6 0.71
283.51 6 1.64* 284.53 6 1.80* 283.15 6 3.36* 284.70 6 2.77* 284.04 6 2.45*
284.70 6 2.59* 285.05 6 2.33* 285.55 6 3.17* 283.80 6 2.79* 285.00 6 2.08*
Data presented as mean 6 SEM. *P < 0.05, significantly different from Sham at the same time-point.
tively using the Odyssey system (LI-COR), such that the ratio of MyoD band density to GAPDH band density (relative optical density, OD) could be calculated using ImageJ (National Institutes of Health, Bethesda, Maryland). The average density ratios of the different groups were compared using statistical analysis. The samples from the Injury, Injury1Stim, and Sham-6 wpi groups were run simultaneously on the same gel. The samples from the Sham-2 wpi and Sham-4 wpi groups were run subsequently on another gel. Statistics. SPSS SamplePower (IBM, Armonk, New York) was used to determine the sample size of each group sufficient for statistical significance with a power of 0.90 (1 – b 5 0.9). All data were distributed normally and are presented as the mean 6 SEM. The data were analyzed using 2-way analysis of variance (ANOVA). When a significant F-ratio was found, a Bonferroni post-hoc test was used to identify the differences. Linear regression analysis was performed to evaluate the relationship between the percentage of eMyHC-positive fibers and normalized peak force / normalized area under the curve (NPF/NAUC) (separately). Differences were considered significant when P < 0.05, and analyses were performed using SPSS, version 16.0 (IBM). RESULTS SFI and Muscle Mass. SFI data are listed in Table 1. Both the Injury and Injury1Stim groups showed an approximately 80% loss of sciatic nerve function, which was significantly greater than in the Sham group. No significant difference in SFI was observed between the Injury and Injury1Stim groups at any time-point. The muscle length and mass data are listed in Table 2. Muscle mass differed between the 3 groups at each time-point, with the highest muscle mass in the Sham group, a lower muscle mass in the Injury1Stim group, and the lowest in the Injury group. Increase in Satellite Cell Differentiation in Stimulated Muscle after Nerve Crush Injury. Typical images showing differentiated (Pax71/MyoD1) satellite cells in muscle cross-sections from the 3 groups MUSCLE & NERVE
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Table 2. Length and mass of muscle samples.
2 wpi 4 wpi 6 wpi
Sham Length (mm) Mass (g)
Injury Length (mm) Mass (g)
Injury1Stim Length (mm) Mass (g)
34.24 6 1.79 2.37 6 0.14 37.05 6 1.96 2.28 6 0.18 35.14 6 1.78 2.47 6 0.09
37.79 6 1.69 1.12 6 0.15* 35.94 6 1.74 1.02 6 0.19* 35.13 6 1.92 0.96 6 0.18*
38.15 6 2.14 1.87 6 0.21*† 36.27 6 2.00 1.64 6 0.16*† 36.87 6 1.51 1.78 6 0.22*†
Data presented as mean 6 SEM. wpi, weeks post-injury. *P
