Effects of eccentric exercise in rehabilitation of phasic and tonic muscles after leg immobilization in rats

Acta Histochemica 116 (2014) 1216–1224

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Effects of eccentric exercise in rehabilitation of phasic and tonic muscles after leg immobilization in rats Anabelle S. Cornachione a,∗ , Letícia O. Cac¸ão-Benedini a , Deise Lucia Chesca b , Edson Z. Martinez c , Ana Claudia Mattiello-Sverzut a,∗ a

Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, School of Medicine of Ribeirão Preto, University of São Paulo, SP, Brazil b Department of Pathology, School of Medicine of Ribeirão Preto, University of São Paulo, SP, Brazil c Department of Social Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 15 June 2014 Received in revised form 6 July 2014 Accepted 7 July 2014 Keywords: Immobilization Skeletal muscle Rats Eccentric training Rehabilitation

a b s t r a c t Eccentric exercise is an essential resource for skeletal muscle rehabilitation following muscle disuse however, abnormalities linked to the tissue recuperation require further research. Our aim was analyze the adaptation ability of rehabilitated muscular tissue in rats during different periods of eccentric training after 10 days of limb immobilization. Twenty-seven Wistar rats were divided into six groups: immobilized 10 days, immobilized and eccentric trained for 10 days, immobilized and eccentric trained for 21 days, and three age-matched control groups. After sacrifice, soleus and plantaris muscles were frozen, cut and stained for general histology using hematoxylin and eosin and Gomori trichrome methods and immunohistochemical methods for fiber typing (mATPase, NADH2-TR), for capillaries (CD31) and intermediate filaments (desmin, vimentin) and high resolution microscopy of resin embedded material. Immobilization resulted in more intense morphological alterations in soleus muscles such as formation of target fibers, nuclear centralization, a reduction in the number of type I fibers, diameter of type I, IIA, IIAD fibers, and capillaries. After 10 days of eccentric training, increases in the nuclear centralization and the number of lobulated fibers were observed. This period was insufficient to reestablish the capillary/fiber (C/F) ratio and distribution of fiber types as that observed in the control group. However, 21 days of rehabilitation allowed the reversal of all morphological and quantitative abnormalities. For the plantaris muscles, 10-days of training restored their basic characteristics. Despite the fact that immobilization affected soleus and plantaris muscles, 10 days of eccentric training was insufficient to restore the morphological characteristics of soleus muscles, which was not the case observed in plantaris muscle. © 2014 Elsevier GmbH. All rights reserved.

Introduction The maintenance of skeletal muscle cytoarchitecture requires a minimum number of repetitious actions. Muscle disuse resulting

Abbreviations: Immob, immobilized group; C, control group; IE(10) , immobilized and rehabilitated by eccentric exercise for 10 days; C(10) , control group of the IE(10) ; IE(21) , immobilized and rehabilitated by eccentric exercise for 21 days; C(21) , control group of the IE(21) ; H.E, hematoxylin-eosin; mATPase, myofibrillar adenosine triphosphatase; NADH2-TR, reduced nicotinamide adenine dinucleotide tetrazolium reductase; I, fiber type I; IIA, fiber type IIA; IIB, fiber type IIB; IIC, fiber type IIC; IID, fiber type IID; C/F, capillary-to-fiber ratio. ∗ Corresponding authors at: Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, School of Medicine of Ribeirão Preto, University of São Paulo, Av Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brazil. E-mail addresses: anabelle [email protected] (A.S. Cornachione), [email protected] (A.C. Mattiello-Sverzut). http://dx.doi.org/10.1016/j.acthis.2014.07.002 0065-1281/© 2014 Elsevier GmbH. All rights reserved.

from use of limb immobilization devices leads to reduced mechanical transduction and a cascade of structural alterations, mainly in skeletal muscles with tonic characteristics such as the soleus (Pette and Staron, 2000). On the contrary, phasic muscles such as the plantaris are less affected. Muscle fiber phenotypes are also modulated by stimuli transduction as in cases of increased/decreased neuromuscular activity and/or mechanical charge (Talmadge, 2000). Previous studies in which the soleus muscle of rats was submitted either to limb immobilization in shortened position (Loughna et al., 1990) or suspension (Stevens et al., 2000; Cornachione et al., 2008) showed that disuse resulted in atrophy and transition of myosin heavy chain (MHC) from slow to fast isoforms, causing an increase in MHCIIa and a reduction of MHCI. Disuse can also cause changes in local blood flow and in the number of capillaries. These alterations are more evident in the

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first week of disuse, but they can persist for up to three weeks. Simultaneously, an increase in intramuscular connective tissue has been observed (Jozsa et al., 1990; Kannus et al., 1998). This process of disuse and connective tissue proliferation is associated with the obliteration of capillaries, which, in turn, initiates a vicious cycle leading to more intramuscular connective tissue and reduced blood flow (Jozsa et al., 1990). Cornachione et al. (2011a) observed a significant reduction in the capillary/fiber (C/F) ratio in the soleus muscle following hypokinesia. Desmin and vimentin are important intermediary filaments (IFs) that help maintain cell architecture and transduce stimuli in both extracellular and intracellular spaces (Cizkova et al., 2009). Vimentin is a typical IF derived from mesenchyme (Franke et al., 1978), which disappears completely during tissue differentiation (Sejersen and Lendahl, 1993). Vimentin is also present in the initial developmental phase of new myoblasts and myotubes, and at a later stage, desmin appears and remains in skeletal muscle throughout the entire life of the animal (Vaittinen et al., 2001). The composition and organization of these IFs can be affected by neuromuscular diseases (Sarnat, 1992) or as a result of experimental procedures such as immobilization (Vater et al., 1992). These proteins seem to be important regeneration markers when muscles are induced to overload or engage in physical activity after a period of disuse. Despite the deleterious structural and functional effects caused by immobilization, this procedure is still commonly used in clinical practice in the initial phases of treating musculoskeletal lesions. It is known that skeletal muscle regeneration occurs during a period of approximately 21 days composed of three sequential phases: (1) an inflammatory reaction characterized by the invasion of macrophages, (2) an activation phase in which satellite cells differentiate and consolidate, and (3) the maturation of recently regenerated myofibrils (Ciciliot and Schiaffino, 2010). During this recovery period, rehabilitation programs can help promote the reversal of lesions caused by primary musculoskeletal damage and secondary disuse and other additional lesions caused by the initial reloading process. Eccentric exercise has been shown to induce muscular regeneration after periods of hypokinesia (Cornachione et al., 2008, 2011a). Eccentric training programs have produced increased tension levels over those found during concentric and isometric contractions (Olson et al., 1972). Some authors argue that stimuli triggered by eccentric contraction lead to increased protein synthesis and hypertrophy (Mayhew et al., 1995). Eccentric exercise is also related to increased tension in the myofilaments, which is determined by reduced recruitment of motor units (Mayhew et al., 1995), a situation that might lead to cellular damage (Evans et al., 1986). Proske and Morgan (2001) reported that cellular lesions appeared during the initial phase of eccentric training, but the tissue was able to adjust and minimize the occurrence of morphological and functional lesions when the stimulus was maintained for a long time. Physical exercise can optimize the post-disuse recovery period. Based on this, our objective was to analyze the adaptation ability of muscle tissue of rats rehabilitated using different periods of eccentric training following 10-days of limb immobilization. The anti-gravitational attitude of quadrupeds favors the constant recruitment of the soleus muscle in maintaining static posture (Roy et al., 1991; Gregor et al., 2006). On the contrary, the plantaris assists in the phasic fast and rhythmical movements of plantar flexion during movement. Our main hypothesis was that only a period of time exceeding 10 days of eccentric training associated with free movement in a cage would favor the complete regeneration of the soleus muscle.

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Materials and methods Animal groups The Animal Research and Care Committee of the Medical School of University of São Paulo approved all experimental protocols for this study. The protocols and procedures of this study were approved by the Ethics in Animal Experimentation Committee of the University of São Paulo Medical School at Ribeirão Preto (Process # 043/2007). The animals had free access to water and food. They were maintained at room temperature with 12-h light/12-h dark cycle with restricted movement of persons. Twenty-seven Wistar rats (81 days old, mean body weight of 319 g) were divided into six groups: (1) immobilized for 10 days (Immob, n = 6); (2) 91-day old control group (C, n = 3); (3) Immobilized and rehabilitated by eccentric exercise for 10 days (IE(10) , n = 6); (4) 101-day-old control group of the IE(10) (C(10) , n = 3); (5) immobilized and rehabilitated by eccentric exercise for 21 days (IE(21) , n = 6); (6) 112-day-old control group of the IE(21) (C(21) , n = 3). Immobilization procedure The animals were immobilized following the model proposed by Coutinho et al. (2002) and Benedini-Elias et al. (2009). The special immobilization apparatus was composed of stainless steel mesh, cotton, impermeable surgical tape, adhesive tape, micropore tape, impermeable surgical tape, viscolycra fabric and a stapler. The lower right limb was immobilized, fixing the knee in extension position and the ankle in maximum plantar flexion (Cornachione et al., 2013). The contralateral (left) lower limb and the upper limbs were free and allowed the animals to move in the cage. The total immobilization period was 10 days. Eccentric training program The eccentric training program was performed on an electrical treadmill running with 16◦ declination (Takekura et al., 2001) and started 24 h after the removal of the immobilization apparatus. After immobilization for 10 days, the rats in groups IE(10) and IE(21) were submitted to a period of 10 days and 21 days running on a declined treadmill, respectively, following the protocol developed by Norman et al. (2000). Using this approach, the training exercise period started with a 10-min daily running session. Every day, an additional 5 min (adaptation) were added until 40 min of training per day was reached, at a speed of 17 m/min. The animals trained for three consecutive days followed by one day without training. This procedure was applied to avoid overtraining. After the experiment was concluded, the animals were weighed and euthanized by an overdose of thiopental anesthetics and the soleus and plantaris muscles were removed. Histology and histoenzymology For histological procedures, a fragment of the ventral portion of each muscle was removed. The fragments were dusted in talcum powder and frozen in liquid nitrogen. Another fragment from the central portion of each muscle was removed for high resolution microscopy and fixed in formol, dehydrated in alcohol, and embedded in Historesin® (Leica Instruments GmbH, Heidelberg, Germany). 5 ␮m-thick transverse frozen sections of soleus and plantaris muscles fragments were cut with a Leica CM 1850 UV cryotome (−25 ◦ C) (Leica Instruments GmbH, Heidelberg, Germany). Sections were stained with Hematoxylin and eosin (H&E) and modified Gomori’s trichrome techniques, and histoenzymological techniques for myofibrillar adenosine triphosphatase (mATPase)

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(pH of 4.3, 4.6 and 9.4 for the soleus and pH of 4.3 and 10.4 for the plantaris) (Cornachione et al., 2011b) and nicotinamide adenine dinucleotide tetrazolium reductase (NADH2-TR). Immunohistochemistry Mouse anti-rat CD31 antibody (1:500 dilution, TLD-3A12, AbD Serotec, Kidlington, Oxfordshire, UK) was used for the immunostaining of capillaries (Cornachione et al., 2011a); monoclonal mouse anti-human desmin (1:100 dilution, Dako, Glostrup, Denmark) and mouse anti-swine vimentin (1:200 dilution, Dako, Glostrup, Denmark) were used for the immunostaining. The technique included a blockade of unspecified connections with 2% BSA (Bovine Serum Albumin, Sigma–Aldrich, St. Louis, MO, USA) and Normal Horse Serum (Vectastain® Universal Elite® ABC Kit, Cat# PK-6200, Vector Laboratories, Burlingame, CA, USA) and in-solution incubation with the primary antibody for 2 h. Incubation continued by the secondary antibody from the Vector kit (Vectastain® Universal Elite® ABC Kit). Later, the sections were incubated with DAB chromogen for 5 minutes and counterstained with hematoxylin. Processing for high-resolution light microscopy After fixation in formol, the soleus and plantaris fragments were dehydrated in an increasing alcohol series (70%, 95%, and 100%). The fragments were immersed in a pre-infiltrated solution for 24 h at 4 ◦ C overnight and embedded in Historesin® (Leica Instruments GmbH, Heidelberg, Germany). After polymerization of the resin, 2.5 ␮m sections were cut with a Sorvall JB4-A microtome (DuPont Company, Newtown, CT, USA), attached to slides, dried at 55–60 ◦ C, and stained with toluidine blue. Morphological and morphometric analyses Qualitative analysis was performed using a Leica DM 2500 light microscope (Leica Microsystems, Frankfurt, Hessen, Germany) on the slides stained by H.E., trichrome and toluidine blue, and also after enzymatic reactions for mATPase and NADH2 -TR activities. For morphometric analysis, QualiView-Atonus software was used (Atonus Engenharia de Sistemas LTDA, São José dos Campos, SP, Brazil). The fiber diameter was obtained in three randomly chosen fields by measuring different types of muscular fibers at a pH of 4.6 for mATPase, considering a minimal number of 200 fibers. Measurements of the different types of fibers were also performed in three randomly chosen incubated slides, at a pH of 4.6 in the reaction to mATPase for both muscles. For the soleus muscle, type I, IIC, IIA, and FTIIAD fibers were counted. For the deep portion of the plantaris muscle, type I, IIC, IIA, IID, and IIB fibers were counted. The analysis of the C/F ratio was performed in five random fields of the processed slides by antibody CD31, and all capillaries and transverse muscle fibers were counted. For statistical analysis of the smaller diameter and the C/F ratio, the linear model of mixed effects was used with a significance level of 5% (˛ = 5%) and a confidence interval of 95% (IC = 95%) using PROC MIXED of the SAS program, version 9.2. Results Morphology The 10-day limb immobilizing procedure caused morphologic alterations in the soleus muscle such as formation of target fibers, lobulated fibers, fiber size variations, and nuclear centralization (Fig. 1). Plantaris muscle did not exhibit important alterations. The slides processed with antibody for desmin confirmed the presence of target fibers, and simultaneously, the slides processed for vimentin displayed negative immunostaining (Fig. 2).

After 10 days of eccentric training, the soleus muscle exhibited fiber size variations, lobulated fibers, nuclear centralization, a basophilic halo, and splitting fibers (Fig. 1G). Target fibers were also observed in this group, but their morphological aspects were smaller when compared to the immobilized group (Fig. 1F, I). However, non-significant morphological alterations were observed in IE(21) for the same muscle (Fig. 1J). High-resolution microscopy The most significant histological finding observed using this technique was the presence of degenerative lesions and vacuolization in the soleus muscle trained by eccentric exercise for 21 days (Fig. 1). In addition, possible scars representing the fusion of areas between two extremities of the same fiber were observed in this muscle (Fig. 1). No significant involvement was observed in the plantaris muscle after disuse or during rehabilitation. Lesser diameter of fiber types Limb immobilization significantly reduced the lesser diameter of type I, IIA, and IIAD fibers of the soleus and type I, IIC, IIA, and IID fibers of the plantaris (C vs Immob, P < 0.05) (Table 1). The eccentric exercise program applied for 10 days increased the lesser diameter of types IIA and IIAD only in the soleus muscle (C(10) vs IE(10) , P > 0.05). The predominant fibers of this muscle, such as those of type I, only reached similar values to the respective control group after 21 days of training (C(21) vs IE(21) , P > 0.05). However, the plantaris muscle presented satisfactory values for the diameter of its predominant fibers (IID) after only a 10-day eccentric training period. It also restored values for IIC and IIA (C(10) vs IE(10) , P = 0.05). However, type I fibers required a longer period (21 days) to obtain values similar to those of the control group (C(21) vs IE(21) , P > 0.05). Distribution of fiber types in soleus and plantaris muscle Immobilization significantly reduced the number of type I fibers in the soleus muscle (C vs Immob, P < 0.05), and at the same time, increased those of type IIC fibers (C vs Immob, P < 0.05) (Fig. 3A). After the immobilization period, 10- and 21-day eccentric exercise programs caused an increase in type I soleus muscle fibers, displaying similar values or above those of the control groups respectively (C(10) vs IE(10) P > 0.05; C(21) vs IE(21) , P < 0.05) (Fig. 3A). In the case of type IIC fibers, also affected by disuse, 21 days of eccentric exercise were required to reduce percentage of type IIC fibers to similar values of the control group (C(21) vs IE(21) , P > 0.05). For the plantaris muscle, no significant alterations were observed in the different types of fibers after disuse and rehabilitation programs (Fig. 3B). C/F ratio of soleus and plantaris muscles The immobilization procedure significantly reduced the C/F ratio in the soleus muscle, but not in the plantaris muscle. Only the group submitted to 21 days of training showed a significant increase in the C/F ratio for the soleus muscle (C(21) vs IE(21) , P > 0.05) (Table 2), and no relevant alterations in the C/F ratio were observed in the plantaris muscle (Table 2). Discussion Disuse prompts a fast and profound transformation in both the architecture and histochemistry of skeletal muscle fibers, mainly in slow muscles such as the postural muscles. In this study, alterations of a degenerative/regenerative nature, such as nuclear centralization and target fiber formation, were observed in the soleus muscle

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Fig. 1. Effects of rehabilitation of the soleus muscle through eccentric exercise for different periods of time. Histological sections stained H&E (left column), toluidine blue (middle column), and histoenzymological reactions for mATPase at pH of 4.6 (right column). (A)–(C) Control group; (D)–(F) Immobilized group; (G)–(I) Immobilized and rehabilitated group by eccentric exercise for 10 days - IE(10) group; (J)–(L) Immobilized and rehabilitated group by eccentric training for 21 days - IE(21) group. (A)–(C) Normal fiber cytoarchitecture (Bar = 43.5 ␮m); (D) Lobulated fibers (thick arrow), centralized nucleus (thin arrow). Target fibers (*) and fiber size variations (Bar = 28.5 ␮m); (E) Cytoplasmic rarefaction with loss of transverse striations () and target fibers (*) (Bar = 28.4 ␮m); (F) Target fibers (*) (Bar = 25 ␮m); (G) Large variation in lobulated fiber sizes (thick arrows) and many centralized nuclei (thin arrows) (Bar = 21.3 ␮m); (H) Nuclear centralization (thin arrow), cytoplasmic rarefaction (arrow head), and vacuole (circle) (Bar = 23.6 ␮m). (I) Target fibers) (*) (Bar = 28.3 ␮m); (J) Polyhedral fibers with peripheral nuclei (Bar = 42.9 ␮m); (K) Fusion areas between two extremities “scar” (empty arrow); (L) Do not show significant alterations (Bar = 43.9 ␮m).

Fig. 2. Immobilized group showing (A) histoenzymological reactions for NADH2 –TR, (B) Immunolocalization of desmin and (C) vimentin. (A) Target fibers (arrows); (B) Positive immunostaining for desmin in target fibers (arrows); (C) Negative immunostaining for vimentin in target fibers (arrows) (Bars = 28.4 ␮m).

of rats submitted to limb immobilization. The maturation of muscular fibers recently repaired by myogenic precursor cells and satellite cells can justify the findings of nuclear centralization (Hawke and Garry, 2001; Itai et al., 2004) despite the fact that these cells have

not been researched here through a specific technique. However, the formation of target fibers can be related to the reduction of mechanical stimuli transduction, both by afferent and efferent mechanical stimuli determined by disuse. Mechanotransduction is

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Table 1 Average measurements of lesser diameter of different types of soleus and plantaris muscles fibers with respect to 95% confidence intervals. Immob

C

IE(10)

Soleus I IIC IIA IIAD

36.0* (32.7–39.4) 29.3 (25.9–32.8) 30.8* (27.4–34.1) 29.1* (25.5–32.8)

47.5(42.8–52.2) 36.6(29.3–43.9) 38.0(33.2–42.8) 39.6(33.1–46.1)

40.5‡ 37.2† 42.2† 39.5†

Plantaris I IIC IIA IID IIB

26.9* (24.4–29.4) 18.7* (13.2–24.2) 27.2* (24.9–29.5) 32.9* (30.6–35.1) 39.3 (36.8–41.7)

36.2(32.8–39.6) 37.1(28.6–45.7) 32.5(29.2–35.8) 38.9(35.7–42.1) 41.9(38.6–45.2)

32.3† , ‡ (29.9–34.7) 32.7† (28.6–36.7) 33.8† (31.5–36.2) 37.7† (35.5–40.0) 40.9‡ (37.7–44.2)

(37.2–43.8) (33.6–40.8) (38.8–45.6) (35.4–43.6)

C(10)

IE(21)

C(21)

46.8(42.1–51.5) 40.5(34.1–46.9) 40.4(35.7–45.2) 55.6(39.2–72.1)

41.9† (38.6–45.3) 36.0† (31.6–40.3) 39.5† (36.1–42.9) 35.9† (30.8–41.1)

45.8(41.1–50.5) 37.0(31.4–42.5) 39.1(34.3–43.8) 36.3(30.8–41.9)

39.3(35.9–42.6) 38.6(26.9–50.2) 34.6(31.3–37.8) 40.9(37.7–44.1) 47.5(43.4–51.7)

36.8† (34.4–39.1) 33.5† (30.7–36.2) 34.7† (32.8–37.0) 38.0† (35.8–40.3) –

36.7(33.3–40.1) 34.2(25.7–42.8) 33.5(30.2–36.8) 37.7(34.5–40.9) 40.0(36.0–44.1)

Immob, group immobilized; C, group control of immobilized group; IE(10) , immobilized and rehabilitated group by eccentric exercise for 10 days; C(10) , control group of group IE(10) ; IE(21) , immobilized and rehabilitated group by eccentric exercise for 21 days; C(21) , control of group IE(21) . * P < 0.05 compared to C. † P < 0.05 compared to Immob. ‡ P < 0.05 compared to C(10) .

an important factor in the regulation of morphology and cellular architecture (Kjaer, 2004). In skeletal muscles, desmin performs an important role in stimuli transduction, both in extracellular and intracellular environments (Kannus et al., 1998; Cizkova et al., 2009). von Fellenberg et al. (2004) reported that the physiopathological process of target formation in muscular fibers can result

from the inhibition or reduction of afferent informational flux originating in mechanoreceptors. Target fibers can be identified using histoenzymological reactions such as mATPase, but target fibers are easily confused with central-core lesions in this technique because both alterations show hypochromic areas in the center of the cells Antibody for

Fig. 3. Percentage measurements of type I, IIC, IIA, and IIAD soleus muscle fibers (A) and I, IIC, IIA, IID, and IIB plantaris muscle fibers (B). *P < 0.05 when compared to C; † P < 0.05 when compared with Immob; ‡ P < 0.05 when compared to C(10) ; P < 0.05 compared to C(21) . Abbreviations: Immob, immobilized group; C, control group of immobilized group; IE(10) , immobilized and rehabilitated group by eccentric exercise for 10 days; C(10) , control group of group IE(10) ; IE(21) , immobilized and rehabilitated group by eccentric training for 21 days; C(21) , control group of group IE(21) .

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Table 2 Means of capillar/fiber ratio of soleus and plantaris muscles with respective to 95% confidence intervals.

Soleus Plantaris

Immob

C

IE(10)

C(10)

IE(21)

C(21)

2.0* (1.9–2.2) 1.5 (1.3–1.7)

2.4(2.2–2.6) 1.7(1.5–1.9)

2.2‡ (2.0–2.3) 1.5 (1.4–1.8)

2.5(2.3–2.7) 1.6(1.5–3.2)

2.3† (2.2–2.5) 1.7† , || (1.6–1.8)

2.4(2.2–2.6) 1.6(1.4–1.8)

Immob, immobilized group; C, control group of immobilized group; IE(10) , immobilized and rehabilitated group by eccentric exercise for 10 days; C(10) , control group of group IE(10) ; IE(21) , immobilized and rehabilitated group by eccentric exercise for 21 days; C(21) , group control of IE(21) . * P < 0.05 compared to C. † P < 0.05 compared to Immob. ‡ P < 0.05 compared to C(10) . || P < 0.05 compared to IE(10) .

desmin in an immunohistochemical techique is able to show a clear difference between these lesions, In central core illness, the cores are characterized by their lack of desmin. Target fibers have an increased desmin concentration, indicating muscular fiber regeneration (von Fellenberg et al., 2004). Another characteristic that differentiates target fibers is the presence of an oxidation activity area in enzymatic reactions such as NADH2 –TR (von Fellenberg et al., 2004). Armand et al. (2003) suggested that morphological variations of the target fiber could be related with evolution or regression of the moth-eaten formation in the cell (Carpenter and Karpati, 2001). In this study, an increase in the enzymatic activities in cell centers was observed with the NADH2 –TR histoenzymological technique. Ten days of eccentric exercise increased the morphological alterations previously induced by immobilization of the soleus muscle. Warren et al. (1994) performed eccentric contractions through electro-stimulation after hyperkinesia in the soleus muscle of mice. They suggested that muscular fiber lesions could be associated with the reduced contractile properties of the fibers. Armand et al. (2003) trained rats with downhill treadmill running and concluded that eccentric exercise accentuates the degeneration/regeneration muscular process. On the other hand, Carpenter and Karpati (2001) stated that target fibers, once formed, could be reversed any time. In this study, it seems that eccentric exercise progressively stimulated the remodeling of muscular fibers since after 10 days of eccentric training, it was possible to observe a reduction of the target lesion of the fibers and after 21 days, the morphological alterations had almost completely disappeared. Proske and Morgan (2001) reported that disrupted sarcomeres in myofibrils and damage to the excitation–contraction (E–C) coupling system are observed in the first week of eccentric stimulation and that the maintenance of the exercise program reduces the muscular damage. The results shown with the immunolabeling for desmin and vimetin suggested an advanced process of cell lesion/regeneration because desmin labeling was positive and vimentin labeling was negative. Peters et al. (2003) observed positive vimentin and negative desmin staining in the anterior tibialis muscle of rats after 24 h of eccentric contractions, using an immunohistochemical technique. Barash et al. (2002) also observed similar results for desmin in the anterior tibialis muscle, up to 24 h (acute phase) after eccentric exercise; however, after 72 h of training, they observed positive protein. Distribution of fiber types After a 10-day period of limb immobilization, the soleus muscle exhibited a significant reduction in the number of type I fibers and an increase in type IIC fibers. Sakakima et al. (2004) showed similar results for the soleus muscle after a 14-day immobilization period with a cast. Several studies have shown that disuse conditions discharge Ca+ in cytosol (Bastide et al., 2000; Shenkman and Nemirovskaya, 2008) by either the influx of calcium from extracellular spaces through existing calcium channels of the membrane, or from leakage of calcium from channels of the

sarcoplasmic reticulum) (Shenkman and Nemirovskaya, 2008). This accumulation of Ca2+ in myofibrils can stimulate responsible pathways by transition from the different fiber types. CalcineurinNFTA1 (NFATC1) seems to be a pathway partially responsible for the change of MHCI (Shenkman and Nemirovskaya, 2008). Dupont-Versteegden et al. (2002) found NFATc1 in the nucleus of soleus muscle fibers after hypokinesia of the lower member. Allen et al. (2001) observed that NFATC1 has the ability to create MHCIIa. Both periods of remobilization (10 and 21 days) resulted in an increase in the number of type I fibers in soleus muscle, where the fiber number was restored (IE(10) ) or even exceeded (IE(21) ) with respect to the control values. The number of type IIC fibers decreased when compared to the immobilized group, and only the group trained for 21 days recuperated the reference values (C(21) ). Sakakima et al. (2004) observed in soleus muscle of Wistar rats a reduction of the number of type II fibers, that were increased by immobilization, after different treadmill running protocols. This study suggests that eccentric exercise may recruit preferable type II fibers, converting them into type I fibers. This, in turn, supports the hypothesis of Nardone and Schieppati (1988) that fast fibers are preferably recruited over slow contracting ones during eccentric contractions. Despite their functional similarities, the soleus and plantaris muscles are morphologically different. The plantaris, by being a dynamic muscle, acts as a supporter in phasic fast and rhythmic movements in plantar flexion during movement. Cornachione et al. (2011b), Fuller et al. (2006) and Roy et al. (1997) observed a predominance of IID fibers in the plantaris muscle of adult rats through histoenzymology, biochemistry, and immunohistochemistry analyses. Disuse resulted in reduced numbers of IIB and IID fibers. It has been reported that the immobilization of muscles composed predominantly of fast fibers determined transition of the MHCs from fast to slow ones (Loughna et al., 1990; Pattullo et al., 1992; Goldspink, 1999).

Lesser diameter Immobilization reduced the mean lesser diameter of type I, IIA, and IIAD fibers in the soleus muscle. This was also observed after a 14-day cast immobilization period or by hypokinesia (Sakakima et al., 2004; Cornachione et al., 2008). The fast degradation of myofibrillar proteins (Jackman and Kandarian, 2004) through different means such as calpain, caspase-3, and the ubiquitinproteosome system (Powers et al., 2005; Gomes et al., 2007; Smuder et al., 2010) may justify the muscular atrophy mechanism. In the soleus muscle, IIA and IIAD fibers had their dimensions restored after a 10-day eccentric training. On the other hand, type I fibers were restored only after 21 days of eccentric training. Other studies on rehabilitation post-immobilization have also indicated that the complete restoration of muscular fiber size was achieved only after a minimal training of three weeks (Egginton et al., 2001; Cornachione et al., 2008). Type IIA and IID fibers of the plantaris

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also had their diameter restored after a 10-day eccentric training period; however, type I fibers required 21 days. C/F ratio The C/F ratio is intrinsically analogous with the level of muscular activity (Desplanches et al., 1990). In this study, the reduction of muscular activity induced by immobilization both significantly and negatively influenced the C/F ratio in the soleus muscle of animals. It is known that slow twitch fibers have a larger number of capillaries than fast twitch fibers (Annex et al., 1998; Cherwek et al., 2000) and that disuse induces a transition from slow to fast twitch, as observed in the present study. Consequently, the reduced C/F ratio can be justified by the reduction in the number of type I fibers. The period of 10 days of eccentric training did not restore the capillary density in the soleus muscle, despite the distribution of type I fibers being similar to that of the control group. After 21 days of training, the C/F ratio recovered to match that of the control values, both for soleus and plantar muscles. Another study developed for our research group observed that eccentric training applied for 21 days after caudal suspension resulted in recovery of C/F values to match the control conditions in the soleus muscle (Cornachione et al., 2008). Vascular endothelial growth factor (VEGF) has been shown to be an important factor promoting angiogenesis, as it stimulates the proliferation and differentiation of endothelial cells. During physical exercise, there is hypoxia and tension in shearing capillary walls (Milkiewicz et al., 2001). As a consequence, there is an increase in the blood flow accompanying the required oxygen (Blomstrand et al., 1997). In the extensor digitorum longus of rats, both hypoxia and a shearing tension increase induced angiogenesis through VEGF expression by myocytes, following physical exercise (Egginton et al., 2001). Many studies have shown an increase in the capillary density of fast muscles after training on a flat treadmill running (Gute et al., 1996; Kannus et al., 1998; Jensen et al., 2004; Waters et al., 2004). The required time to produce an angiogenic response also seem to be modulated by muscular aerobic performance and by the sectional area of transverse fibers (Degens et al., 2006). In this way, the variation between the angiogenic response of soleus and plantar muscles can also be explained by differences in the predominance and size of fiber types that indicate the contraction profile of the muscles. Few studies have been published on the association between different periods of eccentric training and the C/F ratio, mainly using muscle disuse. Conclusion This study has provided evidence supporting the fact that eccentric exercise can be used as a supporting tool in skeletal muscle rehabilitation after disuse. The majority of alterations induced by immobilization such as the distribution of fiber types and sizes as well as capillarization and cytoarchitectural anomalies were reversible, especially after 21 days of training. Despite the fact that the muscles studied here have similar functions, they differ in their biochemical characteristics. Our initial hypothesis was confirmed, as a 10-day eccentric training period was insufficient to allow the complete rehabilitation of the soleus muscle. The morphological characteristics that were achieved in the plantaris muscle in only 10 days were only observed in the soleus muscle after a 21-day training period. Acknowledgements The authors thank Dr Luciano Neder, Department of Pathology, Ribeirão Preto Medicine School, University of São Paulo for

technical assistance. This study was sponsored by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grants n◦ 2007/52506-4, 2007/51715-9 e 2007/52961-3). Ana Claudia Mattiello Sverzyt is the recipient of fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (Grant n◦ 304682/2009-8).

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