The authors have declared that no competing interests exist.
Conceived and designed the experiments: YS SF JY RL PS. Performed the experiments: YS. Analyzed the data: YS SF PS. Contributed reagents/materials/analysis tools: SF PS. Wrote the paper: YS SF PS.
It is well established that unilateral exercise can produce contralateral effects. However, it is unclear whether unilateral exercise that leads to muscle injury and inflammation also affects the homologous contralateral muscles. To test the hypothesis that unilateral muscle injury causes contralateral muscle changes, an experimental rabbit model with unilateral muscle overuse caused by a combination of electrical muscle stimulation and exercise (EMS/E) was used. The soleus and gastrocnemius muscles of both exercised and non-exercised legs were analyzed with enzyme- and immunohistochemical methods after 1, 3 and 6 weeks of repeated EMS/E. After 1 w of unilateral EMS/E there were structural muscle changes such as increased variability in fiber size, fiber splitting, internal myonuclei, necrotic fibers, expression of developmental MyHCs, fibrosis and inflammation in the exercised soleus muscle. Only limited changes were found in the exercised gastrocnemius muscle and in both non-exercised contralateral muscles. After 3 w of EMS/E, muscle fiber changes, presence of developmental MyHCs, inflammation, fibrosis and affections of nerve axons and AChE production were observed bilaterally in both the soleus and gastrocnemius muscles. At 6 w of EMS/E, the severity of these changes significantly increased in the soleus muscles and infiltration of fat was observed bilaterally in both the soleus and the gastrocnemius muscles. The affections of the muscles were in all three experimental groups restricted to focal regions of the muscle samples. We conclude that repetitive unilateral muscle overuse caused by EMS/E overtime leads to both degenerative and regenerative tissue changes and myositis not only in the exercised muscles, but also in the homologous non-exercised muscles of the contralateral leg. Although the mechanism behind the contralateral changes is unclear, we suggest that the nervous system is involved in the cross-transfer effects.
There is a wide range of examples in the literature showing that unilateral intervention produces bilateral effects. The measured effects vary from adaptations of muscle performance to alterations in gene expression, inflammation and tissue remodeling
When properly performed, exercise can provide significant functional benefits that includes increased muscle mass, strength and resistance to fatigue. However, pronounced muscle overuse, and especially that due to unaccustomed eccentric exercise and electrical stimulation, can also induce muscle damage. This injury is generally characterized by changes in fiber morphology and an increased amount of connective tissue, as well as muscle fiber degeneration, necrosis and inflammation
To test the hypothesis that there are bilateral morphological effects in the muscle tissue in response to marked one-sided overuse, we have used an experimental model of unilateral muscle overuse that affects the rabbit triceps surae muscle. The triceps surae consists of a pair of muscles, the soleus and gastrocnemius muscles, which have different molecular, metabolic and physiological characteristics. Both the soleus and gastrocnemius muscles of one leg were exposed to repeated electrical muscle stimulation and exercise (EMS/E) for three experiment periods, 1, 3 and 6 week. The muscles of both the exercised and contralateral non-exercised leg were analyzed with enzyme- and immunohistological methods, and the evaluations were focused on structural changes in the tissue and a possible inflammation.
A total of 24 female New Zealand white rabbits, aged from 6–9 months and having a weight of approximately 4 kg, were used in this experiment. The animals were divided into four groups consisting of six animals in each group. Three groups were exposed to the experimental exercise procedure on their right leg, and one group served as control. All animals were anaesthetized throughout the experiment by intramuscular injections of fentanylfluanison (0.2–0.3 ml/kg) and diazepam (0.2 ml/kg; 5 mg/ml). In order to maintain anesthesia, fentanylfluanison (0.1 ml/kg) was further injected every 30–45 minutes during the experimental procedure. To minimize pain after the experiment, buprenorphine (0.01–0.05 mg/kg), was given subcutaneously.
An experimental model using a kicking machine was used for the achievement of passive flexions and extensions of the right ankle joint. This model, which is a modified form of the model that was used by Backman et al
Muscle specimens of an approximate size of 5×10 mm were taken from the distal portions of the soleus and gastrocnemius muscles of both legs. One specimen from each muscle was directly mounted in an OCT compound (Tissue Tek®, Miles Laboratories, Naperville, IL, USA) on a thin cardboard and rapidly frozen in liquid propane chilled with liquid nitrogen. An additional sample from the corresponding site in the muscles was immediately fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.0, for 24 hours at 4°C. After overnight washing at 4°C in Tyrode’s solution containing 10% sucrose, the chemically fixed samples were mounted and frozen as described above. Both the chemically fixed and the unfixed specimens were stored at −80°C until sectioning for microscopic analyses.
Muscle cross-sections, 7–8 µm thick, were cut in a cryostat microtome at –20°C and mounted on glass slides. The sections were stained with routine hemotoxylin-eosin (H&E) for demonstration of basic histology, including detection of degenerative and regenerative processes, inflammation and muscle fibers containing internal myonuclei
Five µm thick sections, serial to those used for H&E stain, were mounted on chrome-alum gelatin pre-coated glass slides and processed with previously well-characterized monoclonal antibodies (mAbs) against different types of white blood cells, nerve structures, glia cells and proteins that are related to muscle fiber degeneration, regeneration and necrosis. Macrophages were detected by using a monoclonal antibody (mAb) against glycoprotein CD68 (M0814, Dako, Denmark, diluted 1∶25) and neutrophils/T-cells were marked with a mAb against a cell surface antigen that is expressed by a subset of T-cells, thymocytes, and neutrophils (MCA805G, AbD Serotec, Oxford, UK, diluted 1∶100). Eosinophils were labeled with a mAb against eosinophil peroxidase (MAB1087, Chemicon, CA, USA, diluted 1∶100). Axons in nerves were stained with mAb T8660 against β-Tubulin (Sigma-Aldrich, USA, 1∶100) and Schwann cells were stained with mAb S2532 against S-100beta (β-subunit) (Sigma-Aldrich, USA, 1∶100). Motor-endplates in the neuromuscular junctions was marked with mAb MAB303 (Chemicon, CA, USA, diluted 1∶100), against acethylcholinesterase (AChE). Monoclonal Ab D33 (Dako, Denmark, diluted 1∶1000) against desmin and mAb 1891 (Chemicon, CA, USA, diluted 1∶200) against fibronectin were used for the detection of muscle fiber degeneration and fiber necrosis. Regenerative fibers were marked with mAb F1.652 (Developmental studies, Hybridoma Bank, USA, diluted 1∶50) against embryonic MyHC and mAb NCL-MHCn (Novacastra lab, Newcastle, UK, diluted 1∶200) against fetal MyHC. Visualization of the basement of the muscle fibers in the sections stained for developmental proteins were performed by double staining with mAb 5H2 against laminin α-2 chain (Novacastra lab, Newcastle, UK, diluted 1∶1000). All stainings were performed on sections of chemically fixed tissue or on post-fixed sections of unfixed tissue with the exception of mAb D33, mAb 1891, MAB303, F1.652, NCL-MHCn and 5H2 where sections of unfixed tissue were used. Post-fixation of the tissue was performed by incubating the sections for 10 min in 2% paraformaldehyde. The sections were then rinsed in phosphate-buffered saline (PBS).
Immunostaining was performed using standard techniques. The sections were first rinsed in PBS for 3×5 min and then incubated for 20 min in a 1% solution of detergent Triton X-100 (Kebo Lab, Stockholm, Sweden) in 0.01 M PBS, pH 7.2, containing 0.1% sodium azide as preservative. After this procedure the sections were rinsed 3×5 min in PBS and incubated in 5% normal rabbit serum in PBS supplemented with 0.1% bovine serum albumin (BSA) in PBS for 15 min in room temperature. All sections, with the exception of those stained with mAb T8660 against β-Tubulin (c.f. below), were thereafter incubated with the primary antibody diluted to appropriate concentrations in PBS with BSA in humid environment. Incubation proceeded for 60 min at 37°C or overnight at 4°C. After washes in PBS (3×5 min) and another incubation in normal rabbit serum, the sections were incubated with a secondary polyclonal Rabbit Anti-Mouse (TRITC) (nr 0276, Dako, Denmark), diluted 1∶40 in 0.1% BSA in PBS, for 30 min at 37°C. The sections were mounted in Vectashield hard set microscopy mounting medium (Dako, Denmark). In order to visualize the cell nuclei, Vectashield with DAPI was used (Dako, Denmark). For details of the laboratory procedures, see Forsgren et al
The staining procedure for mAb T8660 against β-Tubulin (Sigma, USA) differed from the scheme above. The sections were incubated with 5% normal goat serum and 5% normal horse serum and PBS containing 0.1% sodium-azide, 0.1% BSA and 0.2% Triton X-100 for 1 h at room temperature. They were then incubated with the primary antibody for 2 h at room temperature. After washes with PBS (2×30 min), another incubation in 5% normal goat serum and 5% normal horse serum followed. The sections were thereafter incubated with the secondary goat-anti-mouse antibody (Alexa Fluro 488®, diluted 1∶100) for 1 h at room temperature. After further washes, slides were mounted with coverslips using anti-fading Prolong Gold® with DAPI. For details of the laboratory procedures, see Tse et al
For control of unspecific staining, sections were treated as described above, except that normal serum was used instead of primary antibodies. No specific staining was observed in these control sections. The specificities of the reactions obtained with the used antisera have previously been evaluated
Examination of the stained sections was carried out using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Olympus DP70 digital camera (Olympus Optical Co. GMBH, Hamburg, Germany). Analysis of histological changes was performed on sections stained with routine H&E. All calculations were made blinded to the origin of the samples. Evaluation of cells in the inflammatory infiltrates was made on sections stained with H&E and mAbs M0814, MCA805 and MAB1087. Estimation of fiber degeneration and fiber necrosis was based on fiber morphology and stainings for mAb D33 (desmin) and mAb 1891 (fibronectin). Muscle fibers infiltrated with inflammatory cells and with extensive intracellular reactivity for fibronectin and lack of staining for desmin were classified as necrotic fibers. Fibers with positive staining for developmental MyHCs, mAb A4.951 (embryonic MyHC) and mAb NCL-MHCn (fetal MyHC) and a basophilic staining in H&E were classified as regenerative fibers. Axon injury in the nerve fascicles was determined by negative staining for mAb T8660 and changes in Schwann cells appearances were analyzed on sections stained for mAb S100beta and DAPI.
All data are expressed as means and standard deviations (SD). A two way analysis of variance test (ANOVA) was used for analysis of differences in mean of each analyzed parameter in the three experimental groups and the controls. A multivariate analysis of variance test (MANOVA) was used to examine significant differences between the independent variables in each of the four groups. The test included analysis both within and between the exercised and non-exercised sides. The normality in residuals for each trait was examined and the distribution was normal or approximately normal except for one variable with flatter distribution. All the statistical analysis was performed by using the statistical software SAS/STAT vers, 9.2 (SAS Institute Inc, USA). A p-value <0.05 was considered to be significant.
The study was approved by the ethical committee at Umeå University (protocol A34/07) that complied with national (SFS1988:534; 1988:539) and international (2010/63/EU) guidelines and standards in animal research. The approval was obtained before the start of the study. A licensed breeder had bred all the animals for the sole purpose of being used in animal experiments. All efforts were made to minimize animal suffering.
Our results show that the used experimental model caused severe muscle changes and inflammatory cell infiltration in focal regions of the exercised soleus muscles after 1 w of EMS/E. After 3 w and 6 w of EMS/E, structural tissue changes and myositis of various degrees were observed in restricted regions of the muscle samples in both the exercised and the contralateral non-exercised soleus and gastrocnemius muscles. The histological changes were mainly characterized by a marked fiber size variability, changes in fiber form, increased numbers of fibers with internal nuclei and fibers expressing developmental MyHCs, fiber splitting, fibrosis, and degeneration of axons in the nerve fascicles. A mild to severe accumulation of inflammatory cells was generally observed in the affected areas. Necrotic fibers and infiltration of fat were in some cases seen in the foci of the inflamed and fibrotic areas. The soleus muscle was generally more affected than the gastrocnemius muscle, although there was a large inter-individual variability in the severity of abnormalities within both muscles. The affected areas in all muscles were seen in the restricted regions of the samples (
Muscle sample stained with H&E from the exercised gastrocnemius muscle after 1 w of EMS/E. The figure shows a typical pattern of morphological changes and inflammation in local areas of the muscle tissue (bottom part). (Bar = 50 µm).
In an attempt to quantify the magnitude of muscle changes and inflammatory cell infiltration in each muscle of the different experimental groups, the degrees of alterations in each sample were scored according to the criteria given in
Scores | Variability inFiber size | Fibrosis | Fibers with internal nuclei (%) | Fiber splitting(fibers/mm2) | Necrotic fibers(fibers/mm2) | Inflammation |
0 | Normal | None | <2.5 | <0.025 | <0.025 | None |
1 | Small | Mild | 2.5–10 | 0.025–0.1 | 0.025–0.1 | Mild |
2 | Moderate | Moderate | 10–20 | 0.1–0.4 | 0.1–0.4 | Moderate |
3 | Large | Marked | >20 | >0.4 | >0.4 | Severe |
The parameters used are variability in fiber size (overall existence of small and large sized fibers), fibrosis (abnormal presence of connective tissue), frequency of internal nuclei (percentage of fibers containing internal nuclei), frequency of fiber splitting per unit area (split fibers per mm2 muscle cross-sectional area), frequency of necrotic fibers per unit area (necrotic fibers per mm2 muscle cross-sectional area) and inflammation (degree of inflammatory cell infiltration in the muscle).
Variability in fiber size | Fibrosis | Fibers with internal nuclei | Fiber splitting | Necrotic fibers | Inflammation | |||||||
Exercised | Non-exercised | Exercised | Non-exercised | Exercised | Non-exercised | Exercised | Non-exercised | Exercised | Non-exercised | Exercised | Non-exercised | |
|
||||||||||||
Control | – | 0 | – | 0 | – | 0.5±0.5 | – | 0 | – | 0 | – | 0 |
1 week | 1.4±0.5* | 0.4±0.5 | 1.2±0.8* | 0.6±0.5 | 1.4±1.1* | 1.2±0.4 | 1.2±0.8* | 0.6±0.9 | 1.8±0.8* | 0 | 1.2±0.8* | 0.4±0.5 |
3 week | 1.2±0.4* | 1.0±0.0* |
1.3±0.5* | 0.8±0.4* | 1.3±0.5* | 1.3±0.5* | 0.8±1.0* | 0.8±0.4* | 0.8±1.0* |
0.3±0.5 | 1.3±0.8* | 0.8±0.4* |
6 week | 2.2±0.8*▴△ | 2.8±0.4*▴△ | 2.0±0.9* |
2.0±0.6*▴△ | 2.5±0.5*▴△ | 2.8±0.4*▴△ | 2.2±0.8*▴△ | 2.2±0.8*▴△ | 1.7±1.2* | 1.8±0.8*▴△ | 2.0±0.9* |
1.8±0.8*▴△ |
|
||||||||||||
Control | – | 0.2±0.8 | – | 0 | – | 0.7±0.5 | – | 0 | – | 0 | – | 0 |
1 week | 0.5±0.8 | 0.2±0.4 | 0.5±0.8 | 0.7±0.5 | 1.2±0.4 | 1.0±1.0 | 0.2±0.4 | 0.3±0.8 | 0.3±0.8 | 0.3±0.8 | 0.5±0.8 | 0.3±0.5 |
3 week | 1.8±1.0* |
1.5±0.8* |
1.7±1.0* |
1.2±1.0* | 1.7±1.0* | 1.5±1.2 | 1.5±1.4* |
0.8±1.3 | 1.0±1.5 | 0.8±1.3 | 1.5±1.2* |
0.6±1.2 |
6 week | 1.3±0.8* | 1.3±0.8* |
1.0±.0* | 1.2±1.0* | 1.7±1.0 | 1.5±0.9* | 0.5±0.8 |
1.2±1.1* | 0.3±0.5 | 0.7±0.8 | 0.3±0.5 |
0.8±0.7* |
The values are presented as mean ± SD. Significant differences (p<0.05) are marked; * to controls,
3 w
6 w
6 w
The muscles of the control animals had a muscle morphology characterized by densely packed polygonal fibers of about similar sizes. Only occasional fiber splitting, necrotic fibers, fibers containing developmental MyHCs and inflammatory cells were found. The mean number of fibers with internal nuclei was below 2.5% of the total fiber population.
After 1w of EMS/E, several structural changes were found in the muscle tissue (
Muscle samples from the exercised side (left column, A, C, E) and contrateral non-exercised side (right column, B, D, F) of the soleus muscle after 1 w of EMS/E. The sections are stained with H&E. The left column (exercised side) shows fiber hypertrophy and fiber splitting (arrow) (A), small angular fibers (arrowheads) (C) and existence of internal nuclei (arrow) (C) and inflammatory cell infiltration in the area of a necrotic fiber (asterisks) (E). The right column (non-exercised side) shows occurrence of fiber splitting (arrow) (B), internal nuclei (arrow), fiber hypertrophy (D) and an accumulation of inflammatory cells in the extracellular matrix (arrow) (F). (Bar = 50 µm).
Serial sections from a exercised soleus muscle after 1 w of EMS/E. The sections are stained with H&E (A, D) and for embryonic MyHC (B), fetal MyHC (C), desmin (E) and fibronectin (F). Sections B and C are double stained for Laminin α-2 chain for visualization of the basement membrane of muscle fibers. Figures (A–C) show regenerating fibers (arrows) and a necrotic fiber (asterisks) (A, B). This necrotic fiber is shown in figures (D–F). Note the infiltration of inflammatory cells in the necrotic fiber (D), the lack of reactivity for desmin (E) and the extensive reactivity for fibronectin (F) in this fiber (Bar = 25 µm).
Muscle samples from the exercised (left column, A, C, E, G) and non-exercised (right column, B, D, F, H) soleus muscle after 6 w of EMS/E. The sections are stained with H&E. Note the high variability in fiber size (A–H), the presence of fiber splitting (arrows) (A, B), multiple numbers of internal nuclei (arrows) (C, D), fibrosis and infiltration of inflammatory cells (asterisks) (E, F) and fat infiltration (asterisks) (G–H) in both the exercised and non-exercised muscles. (Bar = 50 µm).
A multivariate analysis of variance test for all parameters showed that the morphological changes were higher for all three experimental groups compared to the controls (p<0.001). The changes were higher in the 6 w group compared both the 3 w and 1 groups (p<0.0004).
After 1w of EMS/E, a few histological changes were observed, although the scores for each parameter did not reach statistical significance. A mild infiltration of inflammatory cells and fiber splitting was locally seen in two of the cases (
The multivariate analysis test showed that the scores of the parameters were significantly higher in each experimental group compared to the controls (p<0.05). The differences was significantly higher at 6 w than at 3 w, which in turn was higher than at 1 w (p<0.001).
The multivariate test revealed higher changes in the scores of the used parameters in the exercised than non-exercised muscles after 1 w of EMS/E (p<0.001). After 3 w and 6 w of EMS/E, the scores increased in the same range for both sides.
The muscle was composed of densely packed polygonal formed muscle fibers of about similar sizes. Splitting fibers, necrotic fibers, fibers containing developmental MyHCs and inflammatory cells were absent or very rare. The mean number of fibers with internal nuclei was below 2.5% of the fiber population.
After 1 w of EMS/E, no parameters differed significantly from those of the controls (see
The multivariate analysis showed that the changes of the parameter scores were higher in the experimental groups at both 3 w and 6 w compared to the controls (p<0.002). At 6w the changes were reduced compared to 3 w (p<0.001).
After 1w of EMS/E, there was no significant difference in any parameters compared to the controls (
The multivariate analysis test for the used parameters showed that the changes were higher at 3w compared to the controls (p = 0.02). At 6 w of EMS/E, the values for each parameter showed a trend against significant changes compared to controls (p = 0.06).
The multivariate test revealed no differences between the exercised and non-exercised sides after 1 w of EMS/E. At 3 w, the values for the used parameters were higher in the exercised side than in the non-exercised side (p = 0.002). After 6 w of EMS/E, the values were lower in the exercised side compared to the non-exercised side (p = 0.02).
After 1 w of EMS/E, the histological appearance within nerve fascicles appeared normal. However, after 3 w, and especially after 6 w of EMS/E, histological changes could be observed in some nerve fascicles located in the affected areas in the soleus and gastrocnemius muscles of both the exercised and non-exercised sides (
Cross-sections of nerves fascicles from the non-exercised side of the soleus muscle after 6w of EMS/E. The sections are stained with H&E (A, C) and for β-Tubulin (mAb T8660) (B, D). Framed region in (B) is inserted in larger magnification (top right). Stars show the corresponding area in (A, B) and (C, D). Note the weak or non-existing β-Tubulin immunoreaction for some axons in (B) and (D) (asterisks, marked with arrow in framed region). In (C), ballooned foamy cell structures are marked (arrows). Note also the fibrotic appearance and the presence of a large number of cell nuclei in the nerve fascicle in (C), especially in the area marked with a triangle. (Original magnification x200).
The immunohistochemical staining with the β-Tubulin antibody (mAb T8660) showed that a subpopulation of axons was unstained in the nerve fascicles with an abnormal morphology in the soleus and gastrocnemius muscles of both legs after 3 and 6w of EMS/E (
Cross-sections of a nerve fascicle from soleus muscle (non-exercised side) after 6w of unilateral EMS/E. The sections are stained with H&E (A), mAb S-100beta (B) and mAb S-100beta and DAPI (C). Note the marked presence of connective tissue and a high number of cell nuclei within the nerve fascicle (A). In (B), mAb S-100beta stains Schwann cells. Figure (C) shows two patterns of stained nuclei, one bluish and one pink, where the bluish nuclei are stained only for DAPI. Some of the nuclei located in cell cytoplasm are devoid of S-100beta reaction (small arrows) whilst others nuclei in the cytoplasm exhibits a S-100beta reaction around the nuclei (arrowheads). The cells with pink nuclei showed immunoreaction in the cytoplasm for mAb S-100beta (large arrows). (Original magnification×200).
The immunohistochemical staining reaction for AChE showed a normal pattern for motor endplates in the neuromuscular junctions in both controls and in the 1w experimental groups. After 3w and 6w of EMS/E, a number of fibers in the affected regions of both legs showed an abnormal AChE staining pattern where the AChE reactivity encircled a large part of the muscle fibers (
Serial sections from a control (A, B) and a non-exercised (C, D) and exercised (E, F) soleus muscle after 6w of EMS/E. The sections are stained with H&E (A, C, E) and for AChE (B, D, F). A typical staining pattern for AChE in motor-endplates is shown in the control muscle (B) (arrows at corresponding locations in A and B). Figures D and F show an atypical AChE staining pattern of muscle fibers. Note the high AChE activity in the regenerating fiber in figure (D) (asterisks) and the AChE reaction on the surface of a fiber with normal morphology (arrow) in (F). A necrotic fiber is marked with an arrowhead in figures (C, D). Star marks similar fiber in the cross-sections. (Bar = 25 µm).
The immunohistochemical analysis showed that cells in the inflamed areas of the muscle cross-sections were stained by the antibodies directed against macrophages (CD68, mAb M0814), neutrophil/T-cells (mAb MCA 805G), and eosinophils (MAB 1085) (
Muscle cross-sections from the non-exercised soleus muscle after 6w of EMS/E (A, B, D–G) and from the exercised side of the gastrocnemius muscle (C). The sections A–C are stained for demonstration of neutrophils/T-lymphocytes (mAb MCA805G). The framed region in (A) is in higher magnification in (B) and the parallel section to the framed region stained for H&E is in the inset (B). The figures show a large number of immuno-reactive cells (arrows) in the connective tissue (A–C). Stars show corresponding muscle fiber in (A) and (B). Figure D show infiltration of inflammatory cells in a necrotic fiber stained with H&E and figure E show the corresponding necrotic fiber (asterix) stained with mAb M0814 against CD68 (macrophages). Figure F show eosinophils (mAb MAB1087) infiltrating a muscle fiber (arrow) In (G), eosinophils (arrows) are stained with H&E in the extracellular matrix of another region of the muscle sample. (Original magnification; A ×100, B–E ×200, F and G x315).
In this study we have used an experimental model to cause unilateral muscle overload in the triceps surae muscle in response to passive flexion/extension movements of the ankle joint combined with an active contraction in the flexion phase via electrical stimulation. The most interesting result of this experiment was that the repetitive unilateral EMS/E caused histological changes and inflammation in the muscle tissue not only in the exercised muscles, but also in the contralateral muscles in the non-exercised leg.
After 1w of EMS/E, the exercised soleus muscle showed several structural alterations in the muscle tissue, whereas only a few changes were found in the exercised gastrocnemius muscle. At this time, there were only limited modifications of the tissue in the homologous contralateral non-exercised muscles. After 3w of EMS/E, significant histological changes of the muscle tissue were found bilaterally in both the soleus and gastrocnemius muscles. The magnitude of these abnormalities increased significantly in both sides from 3w to 6w of EMS/E in the soleus muscles, while the changes in the gastrocnemius muscles at this time were more or less in the same magnitude as in the 3w group. These observations imply that the magnitude of alterations due to EMS/E follows a specific time sequence, that there is a delay in the cross-transfer effects to the contralateral side and that the soleus muscle is more susceptible to influence than the gastrocnemius muscle for this type of experimental exercise. The cause of the more severe abnormalities in the soleus than in the gastrocnemius muscle is unclear, but one explanation might be the characteristic differences between the muscles in fiber phenotype composition, i.e. the soleus muscle is mainly composed of slow twitch MyHCI fibers whereas fast twitch MyHCII fibers predominate in the gastrocnemius muscle
Morphological nerve changes were observed in both exercised and non-exercised soleus and gastrocnemius muscles after 3w and particularly after 6w of EMS/E. A subpopulation of axons in some nerve fascicles in the affected muscle areas was unstained for β-Tubulin, indicating axonal degeneration (
Normally AChE activity is restricted to a small area on the muscle fibers that represents motor-endplates in the neuromuscular junctions. In this study, we observed that after 3w, and especially after 6w of EMS/S, some muscle fibers in the affected areas of both the exercised and non-exercised sides had an abnormal staining pattern for AChE where the fibers were more or less encircled by AChE reactivity (
The bilateral histological muscle changes in the muscle tissue were characterized by focal areas with increased variability in fiber size and fibrosis and within these areas there were, apart from an inflammatory infiltration, a high number of fibers with internal nuclei, fiber splitting and necrotic fibers (
Skeletal muscle is a stable tissue with little turnover of nuclei. However, after injury, e.g. due to extensive physical exercise, skeletal muscle has the ability to go through a rapid and extensive regeneration. The initial event of the regenerative process is fiber injury and necrosis, followed by activation of mononucleated cells i.e. an inflammation. This phase is followed by activation of myogenic cells to proliferate, differentiate and fuse, leading to new fiber formation and reconstruction of damaged tissue
In view of the signs of axonal degeneration, it is possible that the marked presence of small-sized fibers relates to denervation atrophy or to newly formed fibers originating from activated satellite cells
In the exercised soleus muscles, we found a focal and mild accumulation of inflammatory cells in the muscle tissue already after 1w of EMS/E (
The first event that occurs after muscle damage is an invasion at the injury site by inflammatory cells. The inflammatory process is a part of the complex biological response to harmful stimuli. There is also some evidence that the nervous system can be involved in the inflammatory processes. Denervation of joints leads to regression of established rheumatoid arthritis (RA) and protection of development of RA
The inflammatory process is normally time-dependent; neutrophils rapidly invade the muscle tissue, later followed by macrophages, cells known to be phagocytic
It is well known that the type of inflammatory response after muscle injury crucially can influence the outcome of muscle repair, or alternatively, fibrosis
The qualitative similarities in tissue changes and inflammation in the exercised and non-exercised muscles after 3 and 6w of EMS/E show that there is a symmetric process. This observation is in line with previous findings that indicate that there is a signaling system across the midline of the body
The contralateral responses observed in the muscles, including the nerve tissue, could hypothetically be mediated through systemic or circulatory effects. However, since the histological changes and the myositis occurred only focally and not generally in the muscle tissue, it is unlikely that the main part of the changes can be related to a systemic or circulatory effect. Previous studies have also shown that lesion of nociceptive nerves supplying either the contralateral or the ipsilateral limb prior to inflammatory insults, by using surgery, capsaicin, or local anaesthesia, abolished the contralateral responses
It cannot at present be determined to what extent the histological abnormalities in the muscles are a consequence to the use of EMS. It is well known that EMS can have positive effects on muscle regeneration and muscle growth
In conclusion, the present study shows that the repetitive unilateral EMS/E used in this study overtime leads to focal muscle tissue changes in form of muscle fiber affection, including fiber degeneration, fiber regeneration and myositis in both the exercised and the non-exercised sides. Furthermore, we also show that there are bilateral changes in the nerve fascicles and in the reactivity pattern of AChE on fibers. These results extend existing evidence of cross-transfer effects after manipulations of one of the extremities and show that unilateral overuse by using EMS/E can have deleterious effects also on the homologous contralateral muscles. Although the exact mechanisms to the morphological contralateral changes observed in this study are unclear, our results indicate that the cross-transfer effects are mediated by the nervous system. Even if these results may not be directly transferable to the human situation, the findings may be important to consider in a wide range of musculoskeletal and neuromuscular disorders. It is also important to keep this effect in mind when using the contralateral muscle as a control muscle in unilateral exercise experiments.
The authors are grateful to Ms. Ulla Hedlund for excellent technical assistance at the laboratory, to Mr. Adrian Lamouroux and Ms. Fellon Robson-Long for excellent technical service concerning the animal model, Dr. Clas Backman for co-operation on the animal experiments, Professor Harry Wu for statistical advice and Dr. Paul Kingham for sharing antibodies and for valuable comments.