Post-immobilization eccentric training promotes greater hypertrophic and angiogenic responses than passive stretching in muscles of weanling rats.

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Post-immobilization eccentric training promotes greater hypertrophic and angiogenic responses than passive stretching in muscles of weanling rats Priscila Cação Oliveira Benedini-Elias a , Mariana Calvente Morgan a , Anabelle Silva Cornachione a , Edson Z. Martinez b , Ana Claudia Mattiello-Sverzut a,∗ a Skeletal Muscle Structure and Function, Laboratory of Scientific Research, Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil b Department of Social Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil

a r t i c l e

i n f o

Article history: Received 21 July 2013 Received in revised form 23 October 2013 Accepted 24 October 2013 Available online xxx Keywords: Immobilization Morphology Plantaris muscle Soleus muscle Stretching Eccentric exercise Rat

a b s t r a c t This study investigated how different types of remobilization after hind limb immobilization, eccentric exercise and passive static stretching, influenced the adaptive responses of muscles with similar function and fascicle size, but differing in their contractile characteristics. Female Wistar weanling rats (21 days old) were divided into 8 groups: immobilized for 10 days, maintaining the ankle in maximum plantar flexion; immobilized and submitted to eccentric training for 10 or 21 days on a declining treadmill for 40 min; immobilized and submitted to passive stretching for 10 or 21 days for 40 min by maintaining the ankle in maximum dorsiflexion; control of immobilized; and control of 10 or 21 days. The soleus and plantaris muscles were analyzed using fiber distribution, lesser diameter, capillary/fiber ratio, and morphology. Results showed that the immobilization reduced the diameter of all fiber types, caused changes in fiber distribution and decreased the number of transverse capillaries in both muscles. The recovery period of the soleus muscle is longer than that of the plantaris after detraining. Moreover, eccentric training induced greater hypertrophic and angiogenic responses than passive stretching, especially after 21 days of rehabilitation. Both techniques demonstrated positive effects for muscle rehabilitation with the eccentric exercise being more effective. © 2013 Elsevier GmbH. All rights reserved.

Introduction Long periods of segmental immobilization cause numerous health problems in muscle tissue. The incidence of musculoskeletal disorders in children is especially high. In an attempt to reverse this injury process, rehabilitation techniques are used to reestablish the joint range of motion and to activate adaptive properties of the muscle to help restore limb function (Cação-Benedini et al., 2013). The methods used include exercise programs and specific stretching exercises for remobilization of the affected muscle group. However, some questions still need to be resolved. What is the best method for muscle rehabilitation after disuse? What are

∗ Corresponding author at: Estrutura e Função do Músculo Esquelético, Laboratório de Pesquisa Científica, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida dos Bandeirantes, 3900, Ribeirão Preto, SP CEP 14049-900, Brazil. E-mail address: [email protected] (A.C. Mattiello-Sverzut).

the effects on the affected tissue? For how long should the exercise program be applied? In order to investigate these questions, the infancy stage of the animal provides an interesting phase for studies on skeletal muscle tissue, in which important changes occur in the activity and maturation of neuromuscular junctions in response to increased functional demands (Punkt et al., 2004). This phase is characterized by the end of postnatal development in which the animal leaves the nest and begins to explore the environment, between week 2 and 3 after birth (Punkt et al., 2004). Events occur at this stage for the correct adaptation of the muscle tissue, with an increase in thyroid hormone levels, transition from polyneuronal to mononeuronal innervations, and modifications in muscle fiber phenotype (Sullivan et al., 1995; Agbulut et al., 2003; Punkt et al., 2004). In addition, the expression of myosin heavy chain (MHC) isoforms and the energy kinetics of myofibrillar ATPase (mATPase) undergo changes after birth in response to environmental stimulation, with these changes determining the morphological classification of muscle fiber types (Agbulut et al., 2003). Data regarding developing skeletal muscle are relatively scarce in the literature, whereas most studies focus on the implications

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Please cite this article in press as: Benedini-Elias PCO, et al. Post-immobilization eccentric training promotes greater hypertrophic and angiogenic responses than passive stretching in muscles of weanling rats. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.10.008

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of disorders and rehabilitation. Changes in neuromuscular activity (excess or absence of load) influence the phenotypic expression of skeletal muscle fibers, force-generating capacity of muscles, and functional range (Sullivan et al., 1995; Okita et al., 2004) particularly in young individuals. A reduction in the cross-sectional area of fibers and alterations in the amount of connective tissue have been observed in soleus and plantaris muscles of recently weaned rats after a period of limb immobilization (Benedini-Elias et al., 2009). In adult animals, load reductions during limb immobilization cause circulation disorders characterized by a decline in the number of capillaries and changes in fiber size and in the phenotype of myosin isoforms, with specific involvement of slow-contracting fibers of postural muscles, such as the soleus muscle (MattielloSverzut et al., 2006; Silva et al., 2006; Cornachione et al., 2008). Furthermore, alterations in the activity of skeletal muscles directly influence the functional performance (Punkt et al., 2004) and fatigability of the muscle, modify its susceptibility to cell damage (Lieber, 2010), and can cause abnormalities in mechanical properties (Mattiello-Sverzut et al., 2006; Carvalho et al., 2008). Remobilization permits reversal of the damage caused by prolonged disuse by means of therapeutic exercise techniques and stretching protocols. Previous studies have shown that eccentric stimuli are associated with an increase in protein synthesis (Ogilvie et al., 1988; Brussee et al., 1997) through the signaling cascades activated by mitogen-activated protein kinase (MAP) and its receptors and phosphatidylinositol-3 kinase (PI-3K), which result in more effective hypertrophy than other types of contraction (Mayhew et al., 1995; Hornberger and Esser, 2004; Burkholder, 2007). However, relatively few studies have investigated skeletal muscle remobilization during the postnatal period using an eccentric exercise protocol. Sakakima et al. (2004) studying 70-day-old female rats, showed that running on a flat surface three times per week restored muscle properties and the range of motion lost during immobilization. A transition from fast fibers (type IIB) to less fast fibers (types IID and IIA) in the plantaris muscle was observed in young rats submitted to flat running training (Sullivan et al., 1995). Predominant eccentric exercise by downhill running on a treadmill has been shown to increase angiogenesis and to restore muscle capillarization in adult rats after hind limb suspension (Cornachione et al., 2011a). In addition, an increase in the diameter of all fiber types and preferential recruitment of fast fibers, increasing the proportion of type IIA, has been observed in soleus muscle after eccentric training (Cornachione et al., 2008). Passive muscle stretching is a procedure used to restore limited range of motion, which may increase the effectiveness of rehabilitation and reverse the abnormalities observed in previously immobilized muscles (Cação-Benedini et al., 2013). The frequency and duration of mechanical stimulation mediated by stretching are important factors that influence the responses of protein synthesis or degradation and adaptation of muscle connective tissue. The application of stretching exercises three times per week during the period of immobilization has been shown to result in significant ultrastructural alterations such as cell swelling (Gomes et al., 2007), indicating degenerative processes (Mattiello-Sverzut et al., 2006). On the other hand, daily stretching sessions caused fiber hypertrophy in previously immobilized soleus muscles (Okita et al., 2001; Coutinho et al., 2006). Furthermore, a single weekly session (Gomes et al., 2004) or exclusive free movement (Coutinho et al., 2006) was unable to prevent the decrease in muscle mass and in the number of serial sarcomeres. Although weanling muscles possess an unexplored plasticity, eccentric exercise, as a potent physiological stimulus, may increase the functional demand of previously immobilized sedentary muscles. In addition, passive stretching may increase the gain in joint range of motion and restore the elastic and contractile properties of muscle fibers. Therefore, the objective of the present study was to

investigate how different types of remobilization, eccentric exercise and passive stretching, for different periods of time, influence the adaptive responses of skeletal muscles in recently weaned rats after hind limb immobilization.

Materials and methods Animals and experimental procedures The study was approved by the Ethics Committee for Animal Research of the Ribeirão Preto Medical School (Protocol No. 042/2007). Wistar rats (21 days old) were supplied by the central animal house of the Ribeirão Preto Campus, University of São Paulo. The age of the animals at the beginning of the protocols used in the study corresponds to the weaning phase. The rats were kept in groups of four in plastic cages (41 cm×34 cm×16 cm) with free access to pelleted food and water. Adequate procedures for hygiene and cage adaptation were used. The animals were divided into the following groups: immobilized (IG, n = 6), right hind limb in a fully flexed position for 10 days; immobilized and eccentric training for 10 days (IEG(10) , n = 6), immobilized for 10 days and then submitted to eccentric training on a declining treadmill for 10 days; immobilized and eccentric training for 21 days (IEG(21) , n = 6), immobilized for 10 days and then submitted to eccentric training on a declining treadmill for 21 days; immobilized and stretched for 10 days (ISG(10) , n = 6), immobilized for 10 days and then submitted to passive stretching for 10 days; immobilized and stretched for 21 days (IEG(21) , n = 6), immobilized for 10 days and then submitted to passive stretching for 21 days; control of immobilized group (CG, n = 3), animals allowed to move freely in the cage for 10 days; control group of 10 days (CG(10) , n = 3), animals allowed to move freely in the cage for 20 days, and control group of 21 days (CG(21) , n = 3), animals allowed to move freely in the cage for 31 days. The animals were sacrificed on the day after the end of the procedures. The immobilization model proposed by Benedini-Elias et al. (2009) for recently weaned rats was used. This model permits daily adaptation according to the growth of the animals during immobilization and consists of maintaining the tibial-tarsal joint in maximum plantar flexion for a period of 10 consecutive days. The animals were anesthetized by intraperitoneal injection of 4% chloral hydrate. The immobilization device was prepared with number 6 stainless steel mesh, cotton, impermeable surgical tape (Nexcare, 3 M Health Care, St. Paul, MN, USA), micropore tape (3 M Health Care), adhesive tape (3 M Health Care), silver tape (3 M Health Care), visco-lycra fabric strips and a stapler. The upper part was similar to a T-shirt and made of visco-lycra fabric. The lower part, divided into anterior and posterior sections, consisted of stainless steel mesh with the margins wrapped with impermeable surgical tape. The anterior section was also wrapped with cotton lining to protect the anterior surfaces of the immobilized limb and hip. Next, the upper and lower portions of device were joined with staples. After immobilization for 10 days, IEG(10) and IEG(21) animals were submitted to 10 and 21 days of decline treadmill training, respectively, consisting of 3 consecutive days of training followed by 1 day of rest (Lynn and Morgan, 1994; Hayward et al., 1999; Norman et al., 2000; Takekura et al., 2001). The exercise period started with daily running for 10 min, which was increased by 5 min per day until reaching 40 min of training. The average velocity on the treadmill was 17 m/min at a decline of 16◦ (Lynn and Morgan, 1994; Hayward et al., 1999; Takekura et al., 2001). After immobilization, animals of groups ISG(10) and ISG(21) were submitted to passive static stretching for 10 and 21 days, respectively, using the same conditions (days, time and interval) as determined for the treadmill training. Passive stretching of the




soleus and plantaris muscles was performed in the right hind limb of rats previously anesthetized by intraperitoneal injection of 4% chloral hydrate. The ankle joint was fixed for 40 min in maximum dorsal flexion with adhesive tape (Gomes et al., 2007). Preparation and processing of muscle fragments The muscles were removed after sacrifice of the animals with an intraperitoneal injection of excess sodium thiopental (Thiopentax, Cristália, Itapira, São Paulo, Brazil). Briefly, a distal incision was made along the tibia of the right hind limb close to the ankle joint and the soleus and plantaris muscles were removed. Next, the muscle fragments were covered in talcum powder, frozen in liquid nitrogen, and stored at −80 ◦ C until the time for processing. Muscle cross-sections (5 ␮m) were obtained with a Leica CM1850 UV cryostat (Leica Microsystems, Wetzlar, Germany) at −25 ◦ C and collected on glass slides (24 × 32 mm). The slides were processed for staining and histochemistry: hematoxylin–eosin, modified Gomori trichrome stain, and myofibrillar adenosine triphosphate (mATPase, EC 2.1.3.5.7.9.1) in acid and alkaline medium (soleus: pH 9.8, 4.6 and 4.3; plantaris: pH 10.4, 4.7, 4.6 and 4.4). The mATPase method was chosen since it permits demonstration of different fiber types in the same section of the muscle fragment. For immunohistochemistry, anti-rat CD31 antibody (dilution 1:6000) was used to stain muscle capillaries according to the method of Cornachione et al. (2011a). The above procedures were carried out in the Laboratory of Neuropathology, Department of Pathology, FMRP-USP, according to routine procedures.

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Table 1 Body weight of the animals at 21 days old (initial weight) and on the day of sacrifice (final weight). Group

Initial weight (g)

Final weight (g)

CG IG CG(10) IEG(10) ISG(10) CG(21) IEG(21) ISG(21)

51.6 (±2.4) 53.7 (±3.1) 57.3 (±3.5) 54.4 (±3.9) 52 (±4.2) 54.1 (±3.4) 55 (±2.8) 55.1 (±1.4)

118 (±5.3) 77.8 (±5.3)* 218.6 (±19.4) 158.9 (±10.4) 163.7 (±14.2) 239.6 (±12.3) 241.2 (±11.7) 222.2 (±19.9)

Results are reported as the mean and standard deviation. Final body weight: ANOVA, p < 0.05. Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ). * Tukey–Kramer: p < 0.01 compared to CG. Tukey–Kramer: p < 0.01 compared to CG(10) .

from three or more random fields of one fragment per animal were analyzed. Capillary/fiber ratio (C/F) The C/F ratio was obtained by immunostaining with anti-rat CD31 antibody according to Cornachione et al. (2011a). Transverse capillaries and fibers were counted in five random fields (20× objective) of slides prepared from the soleus and plantaris muscles. The C/F ratio was calculated by dividing the total number of capillaries by the number of fibers found in the field.

Analysis Histology Hematoxylin/eosin-stained slides were submitted to histological analysis under a DM 2500 Leica Optical Microscope (Leica Microsystems, Wetzlar, Germany). Morphological alterations of the muscle tissue were evaluated in sections stained with hematoxylin/eosin and modified Gomori trichrome techniques. Morphometric analysis was performed using the QualiView software (Atonus Engenharia de Sistemas Ltda., São José dos Campos, São Paulo, Brazil). The images were captured with a Leica DFC 300FX digital video camera connected to a Leica DM 2500 light microscope and a microcomputer. Fiber type distribution Images of three random fields (40× objective) per animal were collected from fragments processed for myofibrillar ATPase. The fiber types of soleus muscle were classified using slides incubated at pH 9.8, 4.6 and 4.3. It was possible to determine pure fiber types (I, IIA and IID) and intermediate/hybrid fiber types (IIC, IIAC and IIAD). The fiber type I was classified using pH 9.8; the fiber types IIA, IID and IIAD were classified using pH 4.6. The pH value of 4.3 was used to confirm the classification of fiber types IIC and IIAC in soleus muscle. In plantaris muscle, the fiber types were evaluated using slides incubated in pH 10.4, 4.6 and 4.4. The pure fiber types were I, IIA, IID and IIB; the intermediate/hybrid fiber was IIC (in the deep portion of the muscle fragment). At pH value of 4.6, the fiber types I, IIA and IID were classified. The pH values of 4.4 and 10.4 were used to confirm the classification of fiber types IIC and IIB, respectively. The soleus and plantaris muscles were analyzed as described by Cornachione et al. (2011b). Lesser diameter The lesser diameter was measured to prevent possible distortions from imperfect cross-sectioning of the sample. Measurements were obtained from the same material (myofibrillar ATPase) as used for the analysis of fiber distribution. A total of 200 fibers collected

Statistical analysis Body weight data were analyzed by ANOVA, followed by the Tukey–Kramer test, using the BioEstat 4.0 program (Sociedade Civil Mamirauá, Belém, Pará, Brazil) (Ayres et al., 2005). Lesser diameter, fiber distribution and C/F ratio were compared between groups based on a linear mixed-effects model using the PROC MIXED procedure of the SAS 9.2 program (SAS Institute Inc., Cary, NC, USA). A level of significance of 5% (˛ = 0.05) and a 95% confidence interval were adopted. Results Body weight The body weight of the animals was obtained at 21 days of age and when they reached the age corresponding to the group they belonged to. Table 1 shows the mean initial and final body weight of the animals. IG animals showed a delay in body gain after immobilization for 10 days when compared to CG (p < 0.01). Similarly, the body weight of IEG(10) and ISG(10) animals did not reach that of CG(10) (p < 0.01). A similar body weight was observed in animals of IEG(21) and ISG(21) and the respective control group, CG(21) (p < 0.05). Histology Detailed analysis of the soleus muscle showed the presence of round-shaped fibers, a bulky nucleus, few central nuclei, and necrosis in IG animals when compared to CG (Fig. 1). The mean number of central nuclei was almost twice as high in the IEG(10) group compared to ISG(10) (Table 2). Similar numbers of central nuclei were observed in IEG(21) and ISG(21) animals. The number of central nuclei decreased between day 10 and day 21 in the two groups (Table 2). No significant alterations in the plantaris muscle were




Fig. 1. Photomicrographs of the soleus (A–E) and plantaris (F–J) muscles stained with hematoxylin-eosin. (A) and (F) (IG): Round fibers of small size containing bulky nuclei (arrow); (B) and (G) (ISG(10) ): round fibers (asterisk), a large number of central nuclei (arrow), higher reactivity in the soleus muscle; (C) and (H) (IEG(10) ): round fibers (asterisk), a large number of central nuclei (arrow), higher reactivity in the soleus muscle; (D) and (I) (ISG(21) ): round fibers (asterisk), presence of central nuclei (arrow); (E) and (J) (IEG(21) ): round fibers (asterisk), presence of central nuclei (arrow). Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ). Scale bar = 60 ␮m.


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Table 2 Number of morphological alterations in soleus muscle fibers using conventional and histoenzymology staining. Morphological alterations

CG

CG(10)

CG(21)

IG

ISG(10)

IEG(10)

ISG(21)

IEG(21)

Nuclear centralization Lobulated fibers Necrosis Splitting/Fragmentation Basophilic halo

1.7 (±2) 0 1.2 (±0.9) 0.5 (±1) 4 (±1.8)

1 (±1) 0 7 (±3) 0 9.7 (±3.8)

1.5 (±2.4) 0 2 (±1.4) 0 2 (±2)

1 (±2) 0.3 (±0.8) 7.8 (±5) 0.7 (±0.8) 0.7 (±0.8)

117.3 (±403.4) 0.7 (±1.63) 2.2 (±2.4) 0.2 (±0.4) 0.3 (±0.8)

206.3 (±486.8) 1 (±2.4) 2.8 (±2.8) 2.3 (±3.7) 0

81.7 (±157.8) 0 2.8 (±3.1) 0 4.3 (±5.7)

88.2 (±98) 0.3 (±0.8) 1.5 (±1.2) 3 (±5.2) 16 (±12.8)

Results are reported as the mean and standard deviation.Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ).

observed between animals submitted to stretching and eccentric exercise. Lesser diameter of fibers Immobilization reduced the diameter of all fiber types in the soleus and plantaris muscles (p < 0.05) (Table 3). The two rehabilitation techniques applied for 10 or 21 days reversed, although partially, the fiber atrophy observed in soleus and plantaris muscles after immobilization (Table 3). There was no significant difference in lesser fiber diameter of the soleus muscle between IEG(10) and ISG(10) animals (Fig. 2A). After 21 days of rehabilitation, the lesser fiber diameter of IEG(21) and ISG(21) animals was greater than that of the groups treated for 10 days (p < 0.05). The lesser diameter of all soleus fibers, except for fiber type IID, was significantly greater in animals of the IEG(21) group when compared to ISG(21) (p < 0.01) (Table 3). In the plantaris muscle, the lesser diameter of fiber types IID and IIB was greater in IEG(10) animals than in ISG(10) animals (p < 0.02). On the other hand, no significant difference was observed in the lesser diameter of plantaris muscle fibers between IEG(21) and ISG(21) (Fig. 3A). Fiber type distribution Fiber types I (p < 0.0001) and IIAD (p = 0.001) were reduced in IG soleus muscle, whereas fiber types IIA (p < 0.0001) and IIC (p = 0.0007) were increased when compared to CG. No significant difference in soleus fiber type distribution was observed between the IEG(10) and ISG(10) groups (Fig. 2). There was an increase in the quantity of fiber type I in the two groups when compared to IG (p < 0.0001), but the numbers did not reach the values seen in CG(10) (p < 0.0001) (Fig. 2C). The number of fiber type IIC continued to be high as observed in IG. However, the distribution of fiber types IIA and IIAD was similar to that seen in CG(10) (Fig. 2). Additionally, fiber distribution in the soleus muscle was similar in IEG(21) and ISG(21) animals, with the observation of an increase in the number of fiber type I and a reduction in fiber types IIC and IIA when compared to IG (p < 0.0001, p = 0.03 and p < 0.0001, respectively). After 21 days of eccentric training or stretching, the distribution of the different fiber types was similar to that observed in CG(21) (Fig. 2C). In the plantaris muscle, a significant reduction of fiber type IIA and an increase of fiber type IID were observed in IG animals when compared to CG (p < 0.0001) (Fig. 3). Comparison between IEG(10) and ISG(10) only showed a difference in the quantity of fiber types IID and IIC (p < 0.01). The number of fiber type IIA observed in the IEG(10) and ISG(10) groups was the same as that seen in CG(10) (Fig. 3C). After 21 days, comparison of IEG(21) and ISG(21) revealed a significant difference in the quantity of fiber types I and IID and the number of fiber types IIA and IIB was increased in the two groups when compared to IG (p < 0.05). Capillary/fiber ratio (C/F) The immobilization procedure caused a significant reduction of the C/F ratio in the soleus and plantaris muscles (p < 0.0001)

(Table 4). Comparison with IG showed that eccentric training and stretching for 10 or 21 days contributed to increase the C/F ratio in the soleus and plantaris muscles (p < 0.0001) (Fig. 4). No significant difference in the C/F ratio was observed between eccentric exercise and stretching maintained for 10 days. However, 21 days of eccentric exercise was able to produce a greater angiogenic response in soleus and plantaris muscles (p < 0.004 and p < 0.0008, respectively) when compared to the same period of stretching (Table 4 and Fig. 4). Furthermore, a significant increase of the C/F ratio was observed in the plantaris muscle of IEG(21) animals, exceeding the mean value seen in CG(21) (p < 0.003).

Discussion The proposal of this study was to investigate the responses of skeletal muscles in postnatal animals to two rehabilitation techniques that differ in their mechanical and neurophysiological properties, but that can potentially be used to modify the deleterious effects of immobilization. These responses were analyzed after 10 and 21 days of rehabilitation. Morphometric variables were evaluated in the soleus and plantaris muscles. These muscles have similar functions and fascicle size, but differ in their contractile characteristics, i.e., fast and slow twitch fibers (Hodson-Tole and Wakeling, 2010). The most satisfactory results were obtained for muscles of animals submitted to 21 days of eccentric training, which showed values close to those of their respective control group, indicating the restoration of morphological characteristics. In the present study, the growth of the animals was affected during the period of immobilization, which is expected for young animals. These body weight alterations are due to hypoactivity of the caged animal during the period of immobilization, as well as reduced food intake and hind limb disuse. Despite the growth delay, the immobilization device was chosen based on the advantages reported by Benedini-Elias et al. (2009). Similarly, the immobilization procedure has also been shown to affect the growth of adult animals (Ianuzzo et al., 1989; Kasper et al., 1993; Coutinho et al., 2004; Gomes et al., 2004). Thus, it was expected that immobilization would also significantly influence the postnatal growth of these animals. After removal of the immobilization device, the control body weight conditions were only achieved after 21 days of rehabilitation. Therefore, a period longer than 10 days is needed to restore the growth of animals after immobilization. The morphological impact of this procedure on muscle fibers was an increase of nuclear volume observed in both muscles. Wang et al. (2006) also found an increase of 30% in the size of myonuclei measured by the cross-sectional area of the nucleus and a reduction in the number of myonuclei in soleus muscle fibers of animals submitted to caudal suspension for 16 days. The period of 10 days of rehabilitation triggered an intense regenerative response in soleus muscle as demonstrated by the presence of a large number of central nuclei and high tissue reactivity. These findings were more pronounced for animals submitted to eccentric exercise than those submitted to stretching. According to Oustanina et al. (2004), newly regenerated myofibers can be


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Table 3 Mean lesser diameter (␮m) and confidence interval (95%) of the different fiber types in the soleus and plantaris muscles. Fiber type Soleus muscle I IIC IIAC IIA IIAD IID Plantaris muscle I IIC IIA IID IIB

CG

CG(10)

CG(21)

IG

ISG(10)

IEG (10)

ISG(21)

IEG(21)

29.2 (26.9–31.4) 23.7 (20.8–6.5) 23.8 (20.8–26.8) 26 (23.7–28.3) 25.9 (23.4–28.4) 24.8 (21.9–27.6)

36.9 (34.6–39.2) 25.9 (23–28.8) 24.9 (20.9–28.8) 34 (31.7–36.3) 33.3 (30.4–36.2) 31.2 (27.8–34.5)

37.9 (35.6–40.2) 32.3 (29.3–35.2) 35.8 (32.3–39.3) 35.4 (33.1–37.8) 35.8 (32.9–38.6) 36.1 (30.2–41.9)

15* (13.4–16.6) 14.1* (12.3–15.8) 13* (11–15) 15.1* (13.5–16.7) 14.6* (12.2–17) 13.1* (8.1–18)

25.1# , * (23.5–26.7) 22.7# (20.9–24.4) 24.8# (22.9–26.7) 26.4# , * (24.7–28) 26.6# , * (24.8–28.4) 27# , * (25–28.9)

24# , * (22.4–25.6) 23.1# (21.4–24.8) 23.1# (21.3–25) 25.5# , * (23.9–27.1) 25.1# , * (23.3–26.9) 24.4# , * (22.2–26.6)

30.5# , * , 䊉 (28.9–32.1) 26# , * ,䊉 (24.1–27.9) 26.4# , * (24.4–28.5) 29.1# , * , 䊉 (27.4–30.7) 30.5# , * , 䊉 (28.5–32.5) 32.2# , 䊉 (29.5–34.8)

34.7# , * , , (33.1–36.3) 32.6# , , (30.7–34.5) 34.8# , , (32.8–36.7) 35.2# , , (33.6–36.9) 35.1# , , (32.3–37.9) 35.6# , (33–38.3)

27 (24.6–29.5) 19.3 (16.6–22) 19.5 (17.2–21.8) 20.7 (18.4–22.9) 23.8 (21.4–26.1)

28.1 (25.7–30.5) 17.7 (12.7–22.8) 24 (21.7–26.3) 28.4 (26.1–30.7) 29.8 (27.4–32.2)

32.4 (30–34.8) 25.8 (22.6–29.1) 28.8 (26.5–31.1) 32.2 (30–34.5) 34.8 (32.4–37.2)

14.7* (13–16.4) 13.5* (10.2–16.9) 14* (12.4–15.7) 15.6* (14–17.2) 20.7* (19.1–22.4)

23.3# , * (21.6–25) 19.9# (18–21.8) 21.2# (19.5–22.8) 23.3# , * (21.7–24.9) 27# (25.3–28.7)

24.6# , * (22.9–26.3) 22.1# (20.3–23.9) 22.6# (21–22.2) 25.9# ,䊉 (24.3–27.5) 30.3# , 䊉 (28.6–32)

28.3# , * , 䊉 (26.6–29.9) 24.2# , 䊉 (21.6–26.8) 26.4# , 䊉 (24.8–28) 30.8# ,䊉 (29.2–32.4) 36.3# , 䊉 (34.6–37.9)

26.8# , * (25–28.6) 23.3# (21.3–25.3) 25.4# , * , (23.8–27) 31.7# , (30.1–33.3) 37.3# , (35.6–38.9)

Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ). * p < 0.05 compared to the corresponding control group. # p < 0.0001 compared to IG. 䊉 p < 0.05 compared to ISG(10) . p < 0.05 compared to IEG(10) . p < 0.01 compared to ISG(21) .

identified by the presence of central nuclei derived from satellite cells. In this respect, original studies and reviews have highlighted the fundamental role of satellite cells in the regeneration of skeletal muscle (Favier et al., 2008; Yablonka-Reuveni, 2011; Relaix and Zammit, 2012). A reduction in the number of central nuclei was observed when the training period was increased to 21 days, indicating deceleration of protein synthesis and tissue regeneration as also reported by Sakakima et al. (2004). Immobilization for 10 days was associated with a significant decrease in the diameter of all fiber types in the soleus and plantaris muscles. Similar results have been reported for skeletal muscle of very young (Picquet et al., 1998; Sakakima et al., 2004) and adult rats (Gomes et al., 2007; Cornachione et al., 2008, 2013) using immobilization or suspension procedures. Both rehabilitation techniques applied for 10 days reversed, in a similar manner, the fiber atrophy observed in soleus muscles after immobilization. However, in the plantaris muscle, the lesser diameter of fiber types IID and IIB was greater in IEG(10) animals than in ISG(10) animals. On the other hand, eccentric training for 21 days seems to induce marked hypertrophic stimulation in the soleus muscle

since IEG(21) fibers were significantly larger than ISG(21) fibers. It is believed that the functional neuromuscular activity triggered by eccentric exercise applied for 21 days contributes to the higher hypertrophy seen in soleus muscle (Armstrong et al., 1983; Takekura et al., 2001). Additional data support this statement, with the lack of observation of a significant difference in mean lesser diameter of the different muscle fiber types between animals submitted to eccentric exercise and animals of the 21-day control group. In contrast, the lesser diameter of muscle fibers was significantly smaller in animals submitted to stretching when compared to the 21-day control group. Soleus muscle hypertrophy might be due to the higher sensitivity of this muscle to situations of hypoxia during exercise, supported by the oxidative capacity of its fibers. A recent study demonstrated that variations in oxygen levels in the satellite cell niche are important for the quiescence, activation and self-renewal of these cells in muscle in vivo (Liu et al., 2012). In addition, satellite cells play a key role in the production of muscle mass and regeneration (Relaix and Zammit, 2012). In contrast, in the plantaris muscle the lesser diameter of the different fiber types was similar in animals submitted to stretching and eccentric exercise applied for 21 days. In a parallel study conducted at our

Table 4 Mean capillary/fiber ratio (C/F) and confidence interval (95%) in the soleus and plantaris muscles. CG

CG(10)

CG(21)

IG

ISG(10)

IEG(10)

ISG(21)

IEG(21)

Soleus muscle

1.62 (1.4–1.8)

2.14 (2–2.3)

2.14 (2–2.3)

0.71* (0.6–0.8)

1.16# , * (1–1.3)

1.27# , * (1.1–1.4)

1.5# , * , 䊉 (1.4–1.6)

1.74# , * , , (1.6–1.9)

Plantaris muscle

1.0 (0.8–1.1)

1.14 (1–1.3)

1.24 (1.1–1.4)

0.52* (0.4–0.6)

0.88# , * (0.8–1)

0.94# , * (0.8–1)

1.26# , 䊉 (1.1–1.4)

1.52# , * , , (1.4–1.6)

Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ). * p < 0.005 compared to the corresponding control group. # p < 0.0001 compared to IG. 䊉 p < 0.0002 compared to ISG(10) . p < 0.0001 compared to IEG(10) . p < 0.004 compared to ISG(21) .




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Fig. 2. Distribution of different fiber types of soleus muscle. (A) Photomicrographs of the soleus muscles processed to myofibrillar ATPase at pH 9.8. (B) Detailed classification of fiber types I, IIA, IIAC, IIC, IIAD and IID at pH 9.8. (C) Mean percentage of the different fiber types in the soleus muscle. *p < 0.005 compared to the corresponding control group; # p < 0.0001 compared to IG; 䊉 p < 0.0002 compared to ISG(10); † p < 0.0001 compared to IEG(10). Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ). Scale bar = 50 ␮m.

laboratory using the same method but adult animals, we observed no significant difference in the lessser diameter of the different fiber types of soleus or plantaris muscle between the different rehabilitation modalities (eccentric exercise and stretching) applied for 10 and 21 days (Cornachione et al., 2013). Therefore, the plasticity of weanling muscles, which are mainly oxidative muscles, differs from that of adult muscles. The physiological stimulus associated with functional rehabilitation provides better improvement than passive stimulation of sedentary muscles after immobilization.

Variations in capillary numbers in skeletal muscle have been associated with the developmental stage and age of the animal, as well as with alterations in the level of muscle activity as a result of physical exercise or disuse (Kano et al., 2002). The C/F ratio was higher in the soleus muscle than in the plantaris muscle in all groups studied. The immobilization procedure induced a significant reduction of C/F ratio in both soleus and plantaris muscles. On the other hand, studies on adult animals using immobilization or suspension techniques found a reduction of C/F ratio only in soleus muscle,




Fig. 3. Distribution of different fiber types of plantaris muscle. (A) Photomicrographs of the plantaris muscles processed to myofibrillar ATPase at pH 10.4. (B) Detailed classification of fiber types I, IIA, IIC, IID and IIB at pH 10.4. (C) Mean percentage of the different fiber types in the plantaris muscle. *p < 0.005 compared to the corresponding control group; # p < 0.0001 compared to IG; 䊉 p < 0.0002 compared to ISG(10); † p < 0.0001 compared to IEG(10); p < 0.004 compared to ISG(21). Groups: Immobilized (IG); immobilized and submitted to eccentric training for 10 (IEG(10) ) or 21 days (IEG(21) ); immobilized and submitted to passive stretching for 10 (ISG(10) ) or 21 days (ISG(21) ); control of immobilized (CG), and control of 10 (CG(10) ) or 21 days (CG(21) ). Scale bar = 50 ␮m.

but not in plantaris or tibialis anterior muscle (Cornachione et al., 2011a, 2013). Accordingly, the angiogenic response of the plantaris muscle differs in animals at different stages of development (infancy vs adult stage). Analysis of the ISG(10) and IEG(10) groups showed an increase of C/F ratio in soleus and plantaris muscles when compared to the IG group, but no significant difference was observed between these two groups. On the other hand, in animals submitted to rehabilitation for 21 days, comparison of C/F ratio between ISG(21) and IEG(21) showed that eccentric training produced a greater

angiogenic response in soleus and plantaris muscles than stretching. In skeletal muscle, angiogenesis in response to exercise has been attributed to metabolic stimulation and/or the presence of a variety of growth factors such as fibroblast growth factor 2, transforming growth factor ␤, and vascular endothelial growth factor (VEGF) (Brown and Hudlicka, 2003). However, it remains unclear which of these factors trigger or maintain capillary growth. Germani et al. (2003) showed that the VEGF receptors Flk-1 and Flt-1 are expressed by quiescent satellite cells in vivo and are modulated by the degree of ischemia in muscle tissue. Furthermore,




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Fig. 4. Photomicrographs of the soleus (A)–(E) and plantaris (F)–(J) muscles immunolabelled with CD31 antibody. Observe different number of capillaries labeled in soleus and plantaris muscles after 21 days of eccentric exercise (E) and (J) when compared to 21 days of stretching (D) and (I), respectively. Groups: (A) and (F) (IG): Immobilized; (B) and (G) (ISG(10) ): immobilized and submitted to passive stretching for 10 days; (C) and (H) (IEG(10) ): immobilized and submitted to eccentric training for 10 days; (D) and (I) (ISG(21) ): immobilized and submitted to passive stretching for 21 days; (E) and (J) (IEG(21) ): immobilized and submitted to eccentric training for 21 days. Scale bar = 85 ␮m.




VEGF has been shown to be responsible for the increase of Flk-1 phosphorylation and to modulate the function of myoblasts during the muscle regeneration process. Therefore, it is believed that situations of hypoxia and increased local blood flow are important factors that induce angiogenesis and muscle repair (Brown and Hudlicka, 2003; Germani et al., 2003). In conclusion, a longer remobilization period is necessary to obtain satisfactory adaptive responses that reverse the changes triggered by disuse in young animals. As a tonic muscle, the soleus muscle was more affected by the immobilization and rehabilitation procedures than the plantaris muscle, which is a phasic muscle. Taken together, the variables analyzed in this study suggest that remobilization by stretching or eccentric exercise was highly effective. In addition, the combination of a rehabilitation period of 21 days and eccentric exercise, which is characterized by active and functional properties, promoted greater hypertrophic and angiogenic responses in soleus and plantaris muscles. Acknowledgements The authors thank Maria Paula M. Scandar and Ana Maria Anselmi for technical assistance, and Dr. Luciano Neder for providing the research laboratory facilities at the Department of Pathology, FMRP-USP. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant No. 07/52506-4) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas (FAEPA, HCFMRP-USP). P.C.O. Benedini-Elias and A. S. Cornachione were the recipients of postgraduate fellowships from FAPESP (Grant Nos. 07/51717-1 and 07/51715-9, respectively). M. C. Morgan was the recipient of a graduate fellowship from FAPESP (Grant No. 07/52960-7). References Agbulut O, Noirez P, Beaumont F, Butler-Browne G. Myosin heavy chain isoforms in postnatal muscle development of mice. Biol Cell 2003;95:399–406. Armstrong RB, Ogilvie RW, Schwane JA. Eccentric exercise-induced injury to rat skeletal muscle. J Appl Physiol 1983;54:80–93. Ayres M, Ayres M Jr, Ayres D, Santos A. BioEstat 4. 0: aplicações estatísticas nas áreas das ciências biológicas e médicas. 4a ed. Belém: Sociedade Civil Mamirauá; 2005. Benedini-Elias PC, Morgan MC, Gomes AR, Mattiello-Sverzut AC. Changes in postnatal skeletal muscle development induced by alternative immobilization model in female rat. Anat Sci Int 2009;84:218–25. Brown MD, Hudlicka O. Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases. Angiogenesis 2003;6:1–14. Brussee V, Tardif F, Tremblay JP. Muscle fibers of mdx mice are more vulnerable to exercise than those of normal mice. Neuromuscul Disord 1997;7:487–92. Burkholder TJ. Mechanotransduction in skeletal muscle. Front Biosci 2007;12:174–91. Cação-Benedini LO, Ribeiro PG, Gomes ARS, Ywazaki JL, Monte-Raso VV, Prado CM, et al. Remobilization through stretching improves gait recovery in the rat. Acta Histochem 2013;115:460–9. Carvalho LC, Shimano AC, Picado CH. Neuromuscular electric stimulation and manual passive stretching when recovering mechanical properties of immobilized gastrocnemius muscles. Acta Ortop Bras 2008;16:161–4. Cornachione A, Cação-Benedini LO, Shimano MM, Volpon JB, Martinez EZ, Mattiello-Sverzut AC. Morphological comparison of different protocols of skeletal muscle remobilization in rats after hindlimb suspension. Scand J Med Sci Sports 2008;18:453–61.

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