a Universidade Federal do Paraná, Curitiba, PR, Brazil
b Associac¸ão dos Pais e Amigos dos Excepcionais (APAE), Pinhais, PR, Brazil
c Hospital Cardiológico Costantini, Curitiba, PR, Brazil
d Pontifícia Universidade Católica do Paraná, Laboratório de Patologia Experimental, Curitiba, PR, Brazil
Introduction
Muscle injury accounts for 60% of sports injuries.1 Despite its high prevalence, there are few studies on its treatment, which hinders the standardization of physical therapy.2 Among the different forms of treatment, the most commonly recommended are: rest; cryotherapy/compression/elevation; early mobilization; laser; ultrasound; and exercises of active mobilization, passive stretching, and concentric and eccentric strengthening.2–5
Although its biological effects in acute and chronic inflammation are not well understood,2,6–8 therapeutic ultrasound (TUS) has been recommended. Piedade et al.7 assessed the effects ofpulsed application on lacerated gastrocnemius muscle of rats, and observed a significant increase in the number of myotubes in the regeneration zone 14 days after treatment. However, despite such results, the appropriate modulation of the TUS parameters (frequency, intensity, mode, treatment time) is still controversial.
In addition to treatment with electrothermal and phototherapeutic resources, exercise is recommended for the treatment of muscle injury. The most commonly prescribed exercise is muscle stretching.1,5 As with TUS, its benefits on the injured fiber are unclear and it still has a low level of evidence. Nevertheless, Hwang et al.,9 in experimental studies (rats), observed that the passive stretching done during the inflammatory, proliferative, and regenerative phases reduced the muscle fibrosis area by 50%, increasing strength and the number of myofibers when compared with the control group, demonstrating a benefit for muscle regeneration. The best results obtained in the study occurred
when the stretching was initiated on the 14th day afterinjury
It is important to emphasize that both TUS and muscle stretching are indicated for the treatment ofmuscle injury,2,5–9 but there is still no consensus on when and how to prescribe them. Moreover, it is unclear whether the association between these therapeutic modalities can benefit the muscle regeneration mechanism. Thus, this study aimed to evaluate the effects of TUS and/or stretching on morphology after muscle contusion in rats.
It is important to emphasize that both TUS and muscle stretching are indicated for the treatment ofmuscle injury,2,5–9 but there is still no consensus on when and how to prescribe them. Moreover, it is unclear whether the association between these therapeutic modalities can benefit the muscle regeneration mechanism. Thus, this study aimed to evaluate the effects of TUS and/or stretching on morphology after muscle contusion in rats.
Inclusion criteria The study included male rats weighing between 250 and 300 g, which underwent muscle contusion without the presence of fracture.
Sample
distribution The animals included (n = 35) were randomized into five groups: control group (CG = 3); lesion group (LG = 8); lesion + ultrasound group (LUG = 8);
lesion + stretching group (LSG = 8); lesion + ultrasound + stretching group (LUSG = 8). The animals were randomized by drawing. The CG Group was placed three times and the LUG, LSG, LG, and LUSG were placed eight times into a plastic bag for drawing. After the animals were weighed, the drawing was conducted, and the animals were assigned into each group and separated into the cages (Fig. 1).
Protocol for promoting muscle contusion
The animals were anesthetized with ketamine (95 mg/kg) and xylazine (12 mg/kg) intraperitoneally and kept in a prone position with the right paw manually immobilized in knee extension and 90? dorsiflexion of the ankle joint. The lesion was produced in the right gastrocnemius muscle (RGM) using a device consisting of a wooden platform with a hollow aluminum tube, graduated at 5 cm, placed perpendicularly to the platform, as previously described by Minamoto et al.11 (Fig. 2). After the lesion, the absence of tibial fracture was assessed by means of palpation and handling.1 Ultrasound protocol For the application of TUS, a researcher immobilized the mouse while another applied the device. To apply TUS (calibrated), the posterior region of the right paw was shaved and positioned in knee extension and 90? dorsiflexion of the ankle joint, and TUS was applied on the medial belly of the RGM. Gel was the contact medium. The effective radiation area was 1
cm2. The apparatus used was the Sonopulse especial (Ibramed), 1 MHz, pulsed at 100 Hz, 50% cycle, intensity7 of 0.5 W/cm2, and application time of 5 min. TUS was started 72 h after the lesion.
Protocol for stretching the gastrocnemius muscle
Animals were placed in the supine position with the front legs immobilized by a researcher, so that another researcher could perform passive stretching. To this end, the maximum dorsal flexion of the ankle joint was done manually with the knee extended, as described by Mattiello-Sverzut et al.12 The protocol was maintained for 30 s13 during each repetition, with an interval of 30 s, four repetitions,14 five times a week (from Monday to Friday). Stretching was initiated on the 10th day after the lesion.
Chronological presentation of the interventions in the groups Table 1 shows the sequence of interventions in each experimental group. As pulsed ultrasound has anti-inflammatory effects (among others), it was decided to use this feature during the inflammatory phase of the lesion. In turn, stretching was started in the fibroblast proliferation phase.9
Euthanasia and muscle collection The animals of all groups were anesthetized with ketamine (95 mg/kg) and xylazine (12 mg/kg) intraperitoneally for removal of the right and left gastrocnemius muscles using scissors and tweezers. Subsequently, the animals were euthanized with an anesthetic overdose (ketamine and xylazine on the 22th day of the study).
Each muscle was separately weighed on an analytical balance and their length was measured by caliper. The muscles were longitudinally divided in half using a scalpel blade. The lateral portion was discarded and the medial portion was longitudinally divided in half. Half of the samples (lateral) had their distal ends fixed in the resting position with ultrafine needles and were maintained for 3 h in glutaraldehyde (2.5%), then nitric acid (30%) for 48 h, and subsequently stored in glycerol (50%). The lateral half was subjected to routine procedures for assessing the number of sarcomeres in series, as described by Williams and Goldspink.15 The medial halfofthe RGM was fixed in 10% formalin for morphological analysis of cross-sectional area of muscle fibers (CSAMF) and collagen percentage. Left gastrocnemius muscle (LGM) was used as control
The number of sarcomeres was assessed to verify whether the combination of ultrasound and/or stretching could
potentiate the increase in the number of sarcomeres in series after lesion. In turn, the cross sectional area and the percentage of collagen were assessed to verify whether the combination of ultrasound and/or stretching could prevent muscle atrophy and reduce the percentage of connective tissue after muscle lesion. All of these mechanisms may be affected after muscle lesion. Identifying the number of sarcomeres in series
For preparation of histological slides, five fibers were isolated (using ultrafine tip forceps) of each muscle. The fibers were photographed under a light microscope (100× objective, immersion). On each fiber, the number of sarcomeres in series was identified in 100 m; three fields ofdifferent different photomicrographs were used along each muscle fiber, totaling 300 m.
After counting the number of sarcomeres in the 300 m area, the total number of sarcomeres was estimated and correlated with the total length of the muscle, measured with calipers at the day of euthanasia (Fig. 3). Thus, the following equation was used: number of sarcomeres in 300 m, multiplied by muscle length, divided by 300. Therefore, the total sarcomere number and length in each isolated fiber were estimated by correlation between the number of identified sarcomeres and the total length of the muscle.15–18
CSAMF and percentage of type I and III collagen For analysis of the CSAMF, the material was stained with hematoxylin and eosin. The photomicrographs were made using a light microscope at 20× magnification. To measure CSAMF, 100 muscle fibers were randomly selected in the central histological section, using cross-sections of 8 m.19 CSAMF was measured with the image program Pro Plus 4.0, and the unit of measurement adopted was the square micrometer (m2). The final arithmetic mean regarding the 100 measured fibers was calculated (Fig. 4).
For analysis of collagen percentage, the material was stained with sirius red. Photomicrographs were made in a light microscope at 20x magnification for histological slides, using cross-sections of 8 m. The images were analyzed using the software Image Pro Plus 4.0. After calibration, a previously polarized image was chosen by the program to establish a pattern of red and green, which was quantified in all images. This image was considered as a “mask”, which was superimposed upon any other images for identification of certain
colors. After the “mask” was overlaid on each image, the program calculated the percentage of red and green colors, which correspond to the mature (type I) and immature (type III) collagen of the endomysium and perimysial, respectively.20 The results were expressed as percentage of mature collagen (I, red) and immature collagen (III, green), and the sum of both is equal to 100% (Fig. 5).
Analysis of results
To assess normality and homoscedasticity, the Shapiro–Wilk and Levene tests were performed, respectively. Inter- and intra-group comparisons were made by one-way ANOVA post hoc Tukey unequal HSD for parametric values; for nonparametric values, the Kruskal–Wallis test was used. Values were considered significant when p ≤ 0.05. Statistica 7 was used for the statistical analyses.
Results
Body weight A significant increase was observed between the initial and final weight in all groups. Regarding absolute weight, the final body weight in the LG was higher than in the LUSG; the value in the LUG was higher than in the LUSG; and the value in the CG was higher than that in the LSG and in the LUSG. In relative difference, a significant increase was noted between LG and LSG; LG and LUSG; LUG and LSG; and LUG and LUSG (Table 2).
Weight and length of the gastrocnemius muscle
There was no statistically significant difference in the comparison of weight and muscle length between the RGM and the LGM and among the groups (Fig. 6A and B).
Estimated number of sarcomeres in series (ENSS)
In the group comparison, a significant increase was observed in the LUSG between the right and the left side. In the
intergroup comparison, a significant increase of the ENSS in the RGM was observed between the LSG and LUG; LUSG and LUG; and LUSG and CG (Fig. 6C).
Sarcomere length and CSAMF
In the comparison of the right and left sides in the LSG, it was found that sarcomere length was greater in the LGM than in the RGM. In the comparison between groups, an increased length of the sarcomeres of the RGM was observed between LUG and LSG (Fig. 6D). CSAMF of the RGM was greater in the LG when compared with LSG (Fig. 7A).
Analysis of type I (mature) and III (immature) collagens
There was no statistically significant difference in the comparison between RGM and LGM in each experimental group (p > 0.05). In the intergroup analysis of RGM, a significant increase (p < 0.05) was found in the percentages of mature collagen (type I) in LG and LSG (40 ± 12% vs. 24 ± 14%); LG and CG (40 ± 12% vs. 21 ± 9%); LUG and LSG (37 ± 10% vs. 24 ± 14%); LUG and CG (38 ± 10% vs. 21%); and LUSG and CG (36 ± 12% vs. 21 ± 9%) (Fig. 7B).
Discussion
Passive manual stretching increased the number of sarcomeres in series and prevented the increase in type I collagen in injured muscles. However, the ultrasound protocol, regardless of association with stretching, showed no antifibrotic effect in these muscles. In relation to body weight, all groups showed an increase when comparing the initial with the final weight. The absolute final body weight was higher in the CG than in the LSG and in the LUSG; the LG and LUG had greater weight than the LUSG. Therefore, the groups that underwent the highest amount ofstimulation (contusion, ultrasound, and stretching) may have presented a negative interference in absolute body weight gain, since the CG had the highest final body weight. There were no significant differences in muscle weight and length, perhaps due to the short intervention period and the
volume of the stretching protocol, that is, only two weeks, for two minutes each session. Coutinho et al.19 observed an increase muscle length after 40 min stretching for three weeks. Therefore, the intervention period and the volume of stretching contribute to longitudinal muscle growth. However, the measurement of the cross-sectional area of muscle fibers and the number of sarcomeres in series count are the most accurate methods for assessing muscle trophism in parallel and in series, respectively.21
Regarding the number of sarcomeres in series, the LSG presented a higher number, demonstrating the effect of stretching on the sarcomerogenesis of injured muscles. This difference may also be due to the volume of stimulus, as the LUG received only five TUS applications and the LSG received ten stretching sessions.19 The combination of TUS with stretching (LUSG) contributed to the increase in sarcomeres in series when compared with the LUG, demonstrating once again the stretching-stimulated sarcomerogenesis, which was also crucial to the growth of sarcomeres in injured muscles. Furthermore, this outcome was confirmed through the observation that the number of sarcomeres in series in the LUSG was higher than in the CG. Thus, this is the first confirmation of an increase in the number of sarcomeres in series in previously injured muscles. This suggests that the adaptation resulting from the association of TUS with stretching was not only a change in the passive tension of the length of the muscle-tendon unit, being sufficient to induce morphological adaptation of the longitudinal muscle.22
In the LUG, the length of the sarcomeres in the RGM was lower than in the LGM; comparing RGMs, the length ofsarcomeres was lower in the LUG than in the LUSG. These outcomes confirm the sarcomerogenesis observed in the LSG, since, according to Koh,23 skeletal muscle can increase or decrease sarcomeres in series to adjust the optimum length of the
sarcomere in which maximum muscle strength will be produced during contraction. Thus, this mechanism might have occurred in the intragroup LSG and in the comparison with the LUG, since, as increased numbers of sarcomeres in series were observed in the LSG, there was a decrease of the sarcomere length in this group; the reverse pattern was identified in the LUG.
Regarding CSAMF, it was observed that this value was higher in the LG than in the LSG; however, no difference was observed when comparing with the CG. The body weight of the LG increased by 31%, vs. 19% in the LSG, which may have influenced the CSAMF, despite the fact that no increase in muscle weight was observed. In the present study, ultrasound and stretching did not influence CSAMF. Market et al.6 assessed the effects of TUS and low-intensity walking exercise for 20 min on the treadmill (speed 14 m/min) for four days, initiated 24 h after mechanical lesion of the gastrocnemius, and also did not observe an increase in CSAMF. Coutinho et al.19 found that 40-min stretching increased CSAMF in the immobilized and stretched group when compared with the group that was only immobilized; those authors concluded that stretching can prevent muscle atrophy, and its application is relevant in immobilized muscles. As the LG presented a higher CSAMF than the LSG, stretching may have interfered in reducing edema and regenerating the sarcolemma, but it was not sufficient to stimulate radial (transverse) muscle growth, as reported in other studies.24,25 However, to further elucidate the mechanisms involved in changes in muscle trophism, studies analyzing the muscle ultrastructure and regulatory genes are suggested.
The injured muscles and those that did not undergo intervention (ultrasound and/or stretching) showed an increased percentage of type I collagen, demonstrating the fibrotic effect of the lesion. This effect was also observed when ultrasound was applied in isolation and in association with stretching. Conversely, when stretching was performed alone, the proliferation of collagen was lower than that observed in muscles that underwent TUS alone. Therefore, it can be concluded that stretching showed an antifibrotic effect when compared with isolated or combined ultrasound.
Williams et al.26 stated that daily stretching of muscles shortened due to immobilization prevents the deposition of connective tissue in the muscle tissue. The study by Hwang et al.9 corroborated these results. They assessed the effects of stretching on injured muscles and observed its antifibrotic effect. These authors indicated that stretching should be started on the 14th day after lesion. However, in the present study stretching was initiated on the 10th day; despite the early initiation, the fibrinolytic effect was still observed. Jarvinen et al.27 have suggested that the increase intramuscular connective tissue contributes to functional deficits, producing tensile strength, compliance, and muscle stiffness. Stretching programs may contribute to increase muscle extensibility, due to the reorganization of intramuscular connective tissue.28
The LUG had a higher percentage of mature collagen in relation to the LSG and the CG. The increased density of connective tissue, associated with a decreased numbers of sarcomeres, which was found in LUG relative to LSG, is characteristic of shortened skeletal muscle under fibrosis process.9,19,21,26 This demonstrates that ultrasound used alone for only five days has no fibrinolytic effect. Rantanen et al.29 used 3 MHz pulsed TUS at 20%, intensity of 1.5 W/cm2, in stationary technique for 6 min; application was initiated in one group 72 h after the lesion and in the other group, 6 h after the esion. The authors found an increased production of fibroblasts after muscle contusion in rats from the groups that had undergone at least five sessions of pulsed TUS. Piedade et al.,7 started daily applications of 1 MHz pulsed TUS at 50%, intensity of 0.57 W/cm2, for 5 min, through the sliding technique, and observed the early onset of type I collagen fibers four days after gastrocnemius muscle lesion in rats; a better structural arrangement and better alignment of myotubes in formation was observed in the group treated with TUS. These authors suggested that TUS can stimulate early aggregation of this type of collagen.
Although no increase in the percentage of type I collagen (mature) was observed in the present study, the authors did not assess the arrangement, that is, the orientation of the collagen fibers. Thus, it is not possible to predict whether this increase in type I collagen may generate functional deficits. Therefore, future studies should use polarizing microscopy for analysis of birefringence of intramuscular connective tissue28 and range of motion.
The outcomes of this study indicate important clinical perspectives, demonstrating that a passive stretching protocol performed ten days after muscle lesion induced sarcomerogenesis and presented an antifibrotic effect, indicating a possible improvement of skeletal muscle extensibility. Thus, respecting the limitations regarding direct extrapolations of the present results humans, it can be hypothesized that stretching exercises could be prescribed for the treatment of muscles injured by contusion.
Study limitations
This study was limited by the use of an animal model, which produces a lower level of scientific evidence. Therefore, randomized controlled clinical trials should be performed to assess the effects of ultrasound, whether or not associated with stretching, in injured skeletal muscle of humans. The authors recommend the use of electron microscopy to assess sarcomere length in future studies. Furthermore, analyses with molecular biology techniques should be used to investigate the genes involved in the processes of repair and tropism of skeletal muscle, especially resulting from the application of the two therapeutic modalities, ultrasound and stretching. Biomechanical assays for analysis of muscle-tendon unit tension are also important to elucidate the viscoelastic properties in response to ultrasound and/or stretching
Conclusion
The passive stretching protocol induced sarcomerogenesis in injured muscles. Furthermore, stretching alone had an antifibrotic effect, while ultrasound, regardless of association the stretching, was not sufficient to prevent the increase of collagen I in injured muscles.
Conflicts of interest The authors declare no conflicts of interest.
Acknowledgements The authors would like to thank the Federal University of Paraná; the National Council for Scientific and Technological Development (CNPq, Process No. 474303/2011-0 and Process No. 308696/2012-3); the Pontifícia Universidade Católica do Paraná (PUC/PR); and Unibrasil.
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