Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury

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1 Am J Physiol Cell Physiol 279: C1677 C1684, Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury ESTHER E. DUPONT-VERSTEEGDEN, 1,3 RENÉ J. L. MURPHY, 2 JOHN D. HOULÉ, 2 CATHY M. GURLEY, 1,3 AND CHARLOTTE A. PETERSON 1,3 Departments of 1 Geriatrics and 2 Anatomy, University of Arkansas for Medical Sciences, and 3 Central Arkansas Veterans Health Care System, Little Rock, Arkansas Received 14 April 2000; accepted in final form 30 June 2000 Dupont-Versteegden, Esther E., René J. L. Murphy, John D. Houlé, Cathy M. Gurley, and Charlotte A. Peterson. Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury. Am J Physiol Cell Physiol 279: C1677 C1684, We have shown that cycling exercise combined with fetal spinal cord transplantation restored muscle mass reduced as a result of complete transection of the spinal cord. In this study, mechanisms whereby this combined intervention increased the size of atrophied soleus and plantaris muscles were investigated. Rats were divided into five groups (n 4, per group): control, nontransected; spinal cord transected at T10 for 8 wk (Tx); spinal cord transected for 8 wk and exercised for the last 4 wk (TxEx); spinal cord transected for 8 wk with transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk, exercised for the last 4 wk combined with transplantation 4 wk prior to death (TxExTp). Tx soleus and plantaris muscles were decreased in size compared with control. Exercise and transplantation alone did not restore muscle size in soleus, but exercise alone minimized atrophy in plantaris. However, the combination of exercise and transplantation resulted in a significant increase in muscle size in soleus and plantaris compared with transection alone. Furthermore, myofiber nuclear number of soleus was decreased by 40% in Tx and was not affected in TxEx or TxTp but was restored in TxExTp. A strong correlation (r 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not in plantaris muscle, in which myonuclear number did not change with any of the experimental manipulations. 5 -Bromo-2 -deoxyuridine-positive nuclei inside the myofiber membrane were observed in Tx- ExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to be controlled by the insulin-like growth factors (IGF), as IGF-I and IGF-II mrna abundance was decreased in Tx soleus and plantaris, and was not restored with the interventions. These results indicate that, following a relatively long postinjury interval, exercise and transplantation combined restore muscle size. Satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus, but not in plantaris, suggesting that cellular mechanisms regulating muscle size differ between muscles with different fiber type composition. muscle atrophy; satellite cells; insulin-like growth factor; myonuclear number SPINAL CORD INJURY (SCI) is associated with a pronounced loss of mass in muscles distal to the site of injury. Muscle weight and myofiber cross-sectional area (CSA) of muscles with different fiber types is decreased, but muscles composed of mainly type 1 fiber types, which serve as postural muscles, atrophy to the greatest extent (36, 44). Skeletal muscular atrophy is associated with a loss of myofiber nuclei whether the atrophy is induced by spinal cord isolation (6), spinal cord transection (22), space flight (8), hindlimb suspension (19), or denervation (50) (for review see Ref. 7). Most studies have focused on the soleus muscle, which is a slow postural muscle composed of 85 90% type 1 myosin heavy chain (MHC)-expressing myofibers and is very susceptible to disuse atrophy. There is some controversy as to whether myonuclear loss occurs in muscles of all fiber types, but at the very least it appears that myonuclear number during atrophy is more tightly regulated in type 1 compared with type 2 MHC-expressing myofibers (8). We and others have shown that the decrease in myofiber nuclear number upon an atrophy-inducing event occurs rapidly in soleus muscles (8, 22) and is associated with an increase in apoptotic nuclei (4, 22), suggesting that nuclear death is a likely mechanism by which myonuclear number decreases with atrophy. Although it is equivocal as to whether atrophy in the plantaris muscle (composed of mostly type 2 MHC-expressing muscle fibers) was correlated with a loss of myofiber nuclei, hypertrophy of this muscle, induced by functional overload, was associated with an increase in myofiber nuclei (6, 43). This is consistent with a number of studies showing that hypertrophy is associated with an increase in myonuclear number (6, 12, 34, 51). Some studies suggest that proliferation of satellite cells is necessary for hypertrophy to occur (40, 41), but others found that hypertrophy in avian muscle can occur without satellite cell proliferation (33). In a previous Address for reprint requests and other correspondence: C. A. Peterson, Univ. of Arkansas for Medical Sciences, Reynolds Center on Aging, Rm. 3121, 629 South Elm St., Little Rock, AR ( petersoncharlottea@exchange.uams.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /00 $5.00 Copyright 2000 the American Physiological Society C1677

2 C1678 study, we investigated the involvement of satellite cells in the attenuation of atrophy by exercise initiated shortly after SCI. Results indicated that the decreased number of myonuclei in soleus muscle after SCI was not restored by exercise, even though there was a significant increase in muscle fiber CSA (22). This indicates that increases in myofiber size can be accomplished without addition of nuclei and that there is considerable plasticity in the nuclear domain of myofibers. Both insulin-like growth factors (IGF-I and IGF-II) are involved in regulating satellite cell proliferation and differentiation (for review see Ref. 24). They are expressed following muscle damage or degeneration (30) and are required for muscle regeneration to occur, since neutralizing antibodies to IGFs inhibited this process (31, 32). In addition, IGF-I has been implicated in muscle fiber hypertrophy. It was shown that mrna and protein levels of IGF-I increased under hypertrophic conditions (2, 9, 20). Moreover, overexpression of IGF-I was associated with myofiber hypertrophy in transgenic mice (17), and local infusion of IGF-I in adult animals resulted in skeletal muscle hypertrophy (3). In vitro, increased levels of IGF-I resulted in myotube hypertrophy following differentiation (38, 46, 49). However, the involvement of IGF-I in the attenuation of atrophy is controversial. A recent study showed that age-associated atrophy was reduced by overexpression of IGF-I (10); however, Criswell et al. (18) showed that atrophy induced by unloading was not prevented by overexpression of IGF-I in transgenic mice. We have shown that muscular atrophy induced by SCI is ameliorated by two interventions: motor-assisted cycling exercise (to be called exercise) and fetal spinal cord transplantation into the lesion cavity (to be called transplantation) (21, 28, 36). If initiated shortly after SCI (5 days), exercise reduced atrophy in a number of hindlimb muscles of rats when measured after short, intermediate, and long intervals post-sci (21, 28, 36). It has also been shown that step training on a treadmill reduced atrophy in spinal cord-injured cats (42, 44). The beneficial effects of exercise are mediated, at least in part, through reflex activity, as blocking neural activity with a conductance-blocking agent (tetrodotoxin) eliminated the beneficial effects of exercise (37). In addition, the effects of exercise are most beneficial to the soleus muscle, which is also the most severely affected by SCI (36, 44). Transplantation of fetal spinal cord tissue into the lesion site has been used as a means to replace damaged spinal cord tissue and to provide a substratum supportive of axonal regrowth (29), and there is evidence that hindlimb function can be restored following transplantation of fetal (35) or peripheral (14) neural tissue. Fetal tissue transplantation performed acutely was associated with a reduction in soleus muscle atrophy measured 90 days post-sci (28). Thus both exercise and transplantation, initiated acutely after SCI, were successful in maintaining muscle mass and/or preventing atrophy of selected hindlimb muscles. More recently, we showed that exercise in combination with transplantation restores muscle mass following extensive atrophy due to SCI (39). The goal of the present study was to identify possible mechanisms for the attenuation of atrophy in this model by focusing on the role of satellite cells in the restoration of muscle mass and myonuclear number and the involvement of locally expressed IGF. The soleus and plantaris muscles were used in this study to compare the response of muscles with different fiber type compositions. METHODS Animals and experimental protocol. All procedures were performed in accordance with institutional guidelines for the care and use of laboratory animals. Adult female Sprague- Dawley rats ( g) were randomly divided into five groups (n 4, per group): control rats did not undergo a spinal cord transection, were not exercised, and did not receive a fetal tissue implant. All other rats underwent a complete transection of the thoracic (T10) spinal cord by creation of an aspiration lesion 2 3 mm in length while under anesthesia with ketamine (60 mg/kg) and xylazine (10 mg/kg). Following surgery, rats received Penicillin Procaine G and a dextrose saline injection, and for about 2 wk manual expression of the urinary bladder was carried out twice daily until animals regained reflexive voiding. Four weeks after transection of the spinal cord, rats were assigned to the following four groups: rats received no further manipulation (Tx); rats were exercised on a motor-driven bicycle, as described previously (21, 22) and detailed below (TxEx); rats received a fetal spinal cord tissue implant as described (28, 29) (TxTp); or rats received a fetal spinal cord tissue implant and were started on the exercise protocol described below 5 days after the implant (TxExTp). Exercise was performed using a custom built motor-driven cycling apparatus. Rats were suspended horizontally in full body slings and their feet secured to the pedals. Cycling speed was maintained at 45 revolutions per min and each exercise bout consisted of two 30-min exercise periods with a 10-min rest period in between. To measure nuclei that underwent cell division shortly after the interventions started, rats received a continuous release pellet containing 100 mg of 5 -bromo-2 -deoxyuridine (BrdU) (Innovative Research America, Sarasota, FL), constructed to give a dose of mg BrdU g body wt 1 day 1 (13). Pellets were implanted subcutaneously in the subscapular region, and the implantation was done at the same time before death in all groups, which coincided with the day before exercise was started in the exercise groups. Four weeks after interventions (total time postinjury was 8 wk for all groups), animals were killed with an overdose of pentobarbital. Soleus and plantaris muscles were dissected and weighed. Muscles from one leg were embedded in freezing medium and snap frozen at resting length in liquid nitrogen cooled isopentane and stored at 70 C for use in immunohistochemistry, whereas muscles from the other leg were snap frozen in liquid nitrogen and stored at 70 C for Northern analysis. Immunohistochemistry. Cross sections of soleus and plantaris muscles were cut on a cryostat (8 m), air dried, and stored at 20 C. MHC isoform detection was performed as described previously (21, 28). Muscle fiber CSA was determined by using NIH Image and by analyzing a region of 200 fibers (21). Detailed description of changes in fiber type distribution and MHC expression in these animals is reported elsewhere (39).

3 C1679 Dystrophin staining was performed to visualize the sarcolemma. A dystrophin antibody (mouse anti-human dystrophin, NCL-DYS2; Vector, Burlingame, CA) was applied at a 1:4 dilution, followed by a rat anti-mouse IgG1 alkaline phosphatase-conjugated secondary antibody (Pharmigen, San Diego, CA). The alkaline phosphatase substrate kit (Vector) was used to yield a red color for dystrophin staining. To count myofiber nuclei, a Hoechst dye was applied after dystrophin staining. Sections were fixed in 2% paraformaldehyde and Hoechst nuclear dye (Molecular Probes, Eugene, OR) was applied at 1.2 ng/ml for 30 min. Sections were viewed with a fluorescent microscope using an ultraviolet (UV) filter package and photographed. Nuclei within the dystrophin-positive sarcolemma were counted in fibers, and the number of nuclei was expressed per 100 fibers. BrdU incorporation was detected using a BrdU antibody (Boehringer Mannheim, Indianapolis, IN) according to manufacturer s instructions and as described in (22). Briefly, soleus and plantaris muscle sections were rehydrated in PBS and reacted with 0.25% hydrogen peroxide to block endogenous peroxidase activity, after which they were fixed in absolute methanol. Sections were incubated in 2 N HCl for 60 min at 37 C to denature the DNA followed by neutralization in 0.1 M borate buffer at ph 8.5. Muscle sections then were incubated in PBS containing 1.0% Igepal (Sigma, St. Louis, MO) to permeabilize the tissue, and all further washes contained 0.1% Igepal. BrdU antibody was applied at a concentration of 6 8 ng/ l and incubated for 1hatroom temperature. After washing, a secondary rat anti-mouse IgG1 biotinconjugated antibody (Zymed, San Francisco, CA) was applied at 1:100 dilution for 1 h at room temperature. Streptavidinperoxidase was applied followed by diaminobenzidine peroxidase substrate (Vector) for color development. Four areas of 50 fibers from different sites in the muscle were counted for BrdU positivity, and numbers were expressed per 100 fibers. To detect BrdU and dystrophin on the same section, muscle sections were rehydrated in PBS and incubated in 0.25% hydrogen peroxide in PBS to block endogenous peroxidase activity. After washes, dystrophin was detected as described above. Sections were then fixed in methanol, and BrdU staining was performed as described above, but without the blocking step. For BrdU-positive nuclei within the sarcolemma (inside dystrophin stain), the fiber type of those fibers was determined on consecutive sections stained with MHC isoform-specific antibodies. Northern analysis for IGFs. RNA isolation and detection were performed as described (21). Briefly, total RNA was isolated from muscles using the guanidinium thiocyanatephenol-chloroform extraction method as described by Chomczynski and Sacchi (15). Ten micrograms of total RNA were electrophoresed through 1% agarose/2% formaldehyde HEPES/EDTA-buffered gels and separated for 2 3 h at 70 V. RNA was then transferred to a nylon membrane (Zeta-Probe; Bio-Rad, Richmond, CA) using a blotting unit (BIOS, New Haven, CT) and UV cross-linked using a Stratalinker (Stratagene, La Jolla, CA). Membranes were sequentially hybridized with the following probes: IGF-I, IGF-II, and 18S rrna. IGF-I and IGF-II probes have been described previously (45), and 18S rrna probe was an EcoR 1 fragment from 18S RNR* (23). Labeling of the probes was performed using the random prime method (Decaprime II kit, Ambion, Austin, TX). Hybridization was performed according to Church and Gilbert (16). Briefly, filters were hybridized overnight in rotating flasks in a hybridization oven (Robbins Scientific, Sunnyvale, CA) at 65 C in buffer containing 0.5% crystalline grade BSA (Calbiochem, San Diego, CA), 1 mm EDTA, 0.5 M NaPO 4, ph 7.2, and 3% SDS. Filters were washed sequentially in wash solution A (1 mm EDTA, 40 mm NaPO 4,pH 7.2, and 5% SDS), wash solution B (1 mm EDTA, 40 mm NaPO 4, ph 7.2, and 1% SDS), and in postwash (1.6 SSC, 5 mm Tris, ph 8.0, 1 mm EDTA, and 0.1% SDS) at 65 C for 40 min each. Filters were exposed to film, and densitometry was performed on the scanned films using NIH Image. Hybridization with IGF-I and IGF-II probes yielded a number of transcripts of different sizes as described previously (27, 47). The most prominent transcripts for IGF-I (1.8 kb) and for IGF-II (3.8 kb) were analyzed. Density of bands of interest for IGF-I and IGF-II was normalized to density of the bands for 18S rrna and expressed as arbitrary density units. Statistics. To test for statistically significant differences, ANOVA was used; in the case of significant differences, the Tukey multiple comparisons test was applied. Statistical significance was assumed at P RESULTS Exercise and transplantation combined restore muscle size. After spinal cord transection, mean myofiber CSA was decreased 55% and 34% in soleus and plantaris muscles, respectively (Fig. 1). A detailed description of changes in muscle mass, muscle-to-body mass ratio, in MHC distribution, and in oxidative capacity is presented elsewhere (39). Exercise alone (TxEx) increased plantaris CSA to levels not different from control, but in soleus the effect of exercise alone failed to reach significance compared with transection alone (P 0.076). Moreover, fetal tissue transplantation alone (TxTp) did not restore the loss in CSA. However, the combined intervention of exercise and transplantation resulted in an increase of CSA in both muscles compared with muscles from Tx animals. In plantaris, CSA was restored to levels not significantly different from control, whereas CSA in soleus muscle increased 28%, but remained different from control. Therefore, exercise and transplantation combined have a benefi- Fig. 1. Exercise and transplantation combined attenuate the decrease in mean myofiber cross-sectional area (CSA) in muscles of spinal cord transected rats. Mean fiber CSA of soleus (solid bars) and plantaris (open bars) muscles from control (Con), spinal cord transected (Tx), transected and exercised (TxEx), transected and transplantation (TxTp), and transected, exercised and transplantation (TxExTp) rats. Bars represent means SE. *Significant difference from control. # Significant difference from Tx. Significant difference from TxTp (P 0.05).

4 C1680 Fig. 2. Myonuclear number increases in TxExTp soleus muscles. Representative cross sections from control (A), Tx (B), and TxExTp (C) muscles immunoreacted with dystrophin antibody (red) and reacted with Hoechst dye (white). Bar 25 m. cial effect on muscle size even if initiated well after spinal cord transection. Restoration of myofiber nuclear number in soleus is correlated with CSA. To investigate whether the changes in muscle size are associated with changes in the number of myofiber nuclei upon transection and following exercise and/or transplantation, myofiber nuclei of soleus and plantaris muscles were counted. Figure 2 shows representative cross sections of soleus muscles from control (Fig. 2A), Tx (Fig. 2B), and TxExTp (Fig. 2C) rats. The sections were stained with Hoechst dye to identify nuclei and immunoreacted with a dystrophin antibody to visualize the sarcolemma. All nuclei residing within the sarcolemma were considered myofiber nuclei. The results of the myonuclear counts are illustrated in Fig. 3A. In soleus muscle the number of myofiber nuclei decreased 40% with transection, and neither exercise nor transplantation alone restored myofiber nuclear number. However, the combined treatment of exercise and transplantation restored the number of myofiber nuclei in the soleus to a level not significantly different from control. In plantaris muscle there was no change in myofiber nuclear number with any of the experimental manipulations. Since the extent of myofiber nuclear loss and the decrease in CSA were very similar in soleus but very different in plantaris muscle, we examined the correlation between the two variables. Figure 3B shows that there is a strong correlation between myofiber nuclear number and myofiber CSA in soleus (r 0.85) but not in plantaris muscles (r 0.51). Therefore, in soleus muscle only, the combination of exercise and transplantation after transection is associated with an increase in myofiber nuclei, which is strongly correlated with myofiber size. Fusion of satellite cells with muscle fibers provides a mechanism for the restoration of myonuclear number. Since in plantaris the myofiber nuclear number did not change with the experimental procedures, the following experiments were only performed on soleus muscles. To investigate whether satellite cell proliferation and fusion was involved in restoration of myofiber nuclear number with exercise and transplantation combined, rats were implanted with BrdU release pel- Fig. 3. Myofiber nuclear number is restored with exercise and transplantation combined and is correlated with CSA in soleus muscle. A: myonuclear number per 100 fibers of soleus (solid bars) and plantaris (open bars) muscles from control, Tx, TxEx, TxTp, and TxExTp rats. Bars represent means SE. *Significant difference from control. # Significant difference from Tx (P 0.05). B: correlation between myonuclear number per 100 fibers and mean fiber CSA of individual soleus (solid circles) and plantaris (open circles) muscles. Correlation coefficient (r) for both lines is shown and was significant (P 0.05).

5 C1681 Fig. 4. Localization of nuclei that had divided identified by 5 -bromo-2 -deoxyuridine (BrdU) incorporation and the correlation of these nuclei with fiber types. Consecutive sections of soleus muscles from TxExTp rat were immunoreacted with dystrophin (red, A and B) and BrdU (brown, A and B). Myofibers expressing type I MHC (antibody A.4951, C), type 2a MHC (antibody SC.71, D), and type 2x MHC (antibody 212F, E) are shown on consecutive sections. Asterisk indicates same fiber in all sections. Solid arrows, BrdU-positive nuclei within the sarcolemma (inside dystrophin stain); open arrows, BrdU-positive nuclei outside the sarcolemma. Positive nuclei were detected within myofibers and were not correlated to a specific fiber type. Bar in E for A, C, D, and E is 25 m; bar in B is 25 m. lets. BrdU incorporation identified cells that have undergone S-phase of the cell cycle, and the nuclei from these cells were followed by immunoreacting muscle sections with a BrdU antibody. BrdU-positive myonuclei located within the sarcolemma, identified by dystrophin staining, were assumed to be from satellite cells that have divided and fused into the existing myofibers. The number of BrdU-positive nuclei in the TxExTp group appeared higher [control ; Tx ; TxEx ; TxTp ; TxExTp (BrdU-positive nuclei per 100 fibers; mean SE)], although the difference between groups failed to reach significance because of the very high variability in the TxExTp group. Therefore, we investigated the two highest labeled soleus muscles for evidence of fusion of satellite cells into muscle fibers. As illustrated in Fig. 4, A and B, numerous BrdU labeled nuclei were observed both outside (white arrow) and inside the sarcolemma (black arrows), indicating that satellite cells had divided and frequently fused into the myofibers in TxExTp muscles. As satellite cells appeared to contribute to restoration of myofiber nuclear number with exercise and transplantation in spinal cord transected rats, we next studied whether satellite cell fusion was restricted to particular fiber types. Consecutive sections were immunoreacted with antibodies to the different MHC isoforms that are present within the soleus muscles. Figure 4, C E, illustrates the MHC expression pattern compared with BrdU incorporation (Fig. 4, A and B) on consecutive sections. BrdU-positive nuclei were observed in myofibers independent of MHC accumulation, suggesting that satellite cells did not preferentially fuse into myofibers of a specific fiber type. IGF-I and IGF-II transcript levels are not correlated with late changes in muscle size. IGF-I and IGF-II mrna abundance was assessed to investigate whether these growth factors are involved in the activation and proliferation of satellite cells and in the response to combined exercise and transplantation. Figure 5 and Table 1 show the results of these experiments. In both soleus and plantaris muscles, IGF-I transcript levels decreased significantly with spinal cord transection and were not restored with exercise or transplantation alone or with the combined intervention. There was no difference in IGF-II mrna abundance in soleus or plantaris muscle with any of the experimental proce- Fig. 5. mrna abundance for insulin-like growth factor I (IGF-I), but not IGF-II, decreases with transection and is not restored with combined exercise and transplantation treatment. Total RNA from soleus and plantaris muscles was analyzed by Northern blotting with indicated probes. All samples were run on one gel, and a composite of representative samples of each probe is shown for the different groups: 1 control, 2 Tx, 3 TxEx, 4 TxTp, and 5 TxExTp. Density of bands was normalized to density of 18S rrna probe, and data are summarized in Table 1.

6 C1682 Table 1. Effect of transection, exercise, and/or transplantation on IGF RNA abundance Group IGF-I IGF-II IGF-I IGF-II Con Tx * * TxEx * * TxTp * * TxExTp * * Values are means SE and are expressed in arbitrary density units. IGF, insulin-like growth factor; Con, control not transected; Tx, transected; TxEx, transected and exercised; TxTp, transected and transplanted; TxExTp, transected, exercised and transplanted. * Significant difference from control. dures. Thus, at the time point measured in this study, IGF mrna levels are not correlated with muscle mass. DISCUSSION Soleus Plantaris Restoration of muscle size. A dramatic loss of mass in muscles distal to the level of injury is associated with SCI. In previous studies it was shown that exercise or transplantation alone attenuated this atrophy if implemented shortly after the injury (21, 28, 36). We show here that with a long postinjury interval prior to intervention, exercise or transplantation alone did not restore muscle size in soleus and plantaris muscle but that the combination of exercise and transplantation restored muscle size in both muscles. This indicates that, even though the extent of atrophy is similar at 10 days post-sci (21) and at 30 days post-sci (36), the reaction of the muscle tissue to interventions is different. Some studies report beneficial effects of exercise even if started after a delayed period following transection, but these exercise regimens were performed for extended periods of time (42, 44). We chose the time point reported here, as we expected to see a restoration in size of soleus muscle with exercise alone. Based on our previous studies, very short (5 days) periods of exercise or a longer period comparable to the one reported here yielded an increase in muscle size if implemented shortly following transection (21, 36). There may be a window of time in which it is most beneficial to start the exercise and there may be a minimal duration of exercise required if started well after injury to show atrophy-reducing effects. Transplantation alone has been shown to be beneficial in attenuating atrophy if performed early after the injury in both soleus (28) and tibialis anterior muscles (36). However, atrophy-reducing effects in soleus were only seen after a long period (90 days) following transplantation and not after 4 wk (36). It is possible that it takes more time for the transplants to restore muscle mass, particularly if the effects of a transplant depend upon axon growth into the graft tissue or for synaptic connections to be made. The mechanisms underlying the increase in muscle size with the combined treatment, but not with either treatment by itself, are unclear. It is possible that the transplants release some factor(s) that requires muscle activity to exert their action, or alternatively, there could be factors released from exercising muscles that are involved in neuronal outgrowth. Neurotrophin-4 is a possible candidate, since its expression increases with increased muscle activity, its expression is greater in type 1 than type 2 muscle fibers, and it mediates nerve sprouting (25). Future studies need to be directed toward investigating the response of this and other activity-dependent neurotrophic factors. Changes in myonuclear number. It has been reported that the decrease in soleus muscle size is associated with a decrease in myofiber nuclei such that the nuclear domain (DNA-to-cytoplasmic ratio) remains relatively constant, irrespective of how atrophy was induced (6, 7, 8, 19, 50). Even though we did not measure the actual size of nuclear domains, but instead myonuclear number and CSA, the same trend was observed in this study. Myofiber nuclear number was very closely associated with muscle size as measured by myofiber CSA. The decrease in myofiber nuclear number associated with transection was restored with the combined treatment of exercise and transplantation as was muscle size. In a previous study we showed that myofiber nuclear number was not restored by short-term exercise even though loss in fiber size was attenuated (21). This indicates that even in soleus, the ratio of myofiber nuclei to CSA is not constant and suggests that restoration of nuclear domain size by addition of nuclei may lag behind restoration of myofiber size. For plantaris muscles the concept of a constant nuclear domain has not been investigated in great detail. In one study it was reported that nuclear domain remained constant in plantaris muscles under hypertrophying conditions (43). The results from our current study indicate that the size of plantaris muscles can vary considerably without a change in myonuclear number, and therefore the size of the nuclear domain in plantaris muscles is not as regulated as appears to be the case for soleus muscles. Satellite cell involvement in restoration of muscle size. The current understanding of the process by which myofiber nuclei are added to myofibers is that activated satellite cells proliferate and fuse into existing fibers (1, 11). It has been suggested that proliferation of satellite cells is required for hypertrophy to occur, since irradiated muscles lose the capacity to increase in size (40, 41). Whether satellite cells also are involved in the restoration of muscle size after atrophy is less clear. It has been shown that attenuation of atrophy by IGF/growth hormone treatment combined with resistance exercise during hindlimb suspension was associated with a decrease in myofiber nuclear loss (5). This could have been accomplished by either a decrease in the loss of nuclei or addition of new nuclei. Indeed, it was shown that resistance exercise during hindlimb suspension decreased apoptotic nuclear loss (4), the mechanism thought to be responsible for the loss of nuclei during atrophy. However, in the current investigation myofiber nuclear loss had already occurred by the time the interventions were started, suggesting that to restore myofiber nuclear number, satellite cells must be involved. Indeed, BrdU-positive

7 C1683 nuclei were observed inside the sarcolemma of TxExTp soleus muscles, indicating that fusion of satellite cells had occurred in muscles with an increased myonuclear number. Since myofiber nuclear number is higher in type 1 than type 2 MHC-expressing myofibers (6, 26, 48), we investigated whether satellite cells fused preferentially with myofibers expressing MHC type 1. In soleus muscles of the experimental animals, a switching of MHC isoforms to a faster phenotype occurs during the experimental manipulations described here (39), and there are only a few fibers that continue to express only one MHC. Nonetheless, BrdU-positive nuclei were observed in fibers expressing all MHCs, arguing against preference for fusion of satellite cells to a particular fiber type. It is interesting that myofiber nuclear number was restored in soleus muscles even though the fiber type of most of the myofibers had changed from slow, MHC type 1, to fast, MHC type 2 (39), which in normal muscle have a lower myonuclear number. In plantaris muscles consisting primarily of type 2 MHC-expressing myofibers, no change was observed in myofiber nuclear number even though considerable changes in muscle size occurred. This observation requires further investigation. IGF involvement in controlling muscle size. The possible involvement of locally expressed IGF-I and IGF-II in the restoration of muscle size and myofiber nuclear number also was investigated in this study. IGF-I and IGF-II are growth factors that contribute to satellite cell proliferation and differentiation in vivo and in vitro (24), and IGF-I has been implicated in skeletal muscle hypertrophy (3, 17, 38, 46). Therefore, we hypothesized that IGF-I would be increased with restoration of muscle size in TxExTp muscles. The results indicate that IGF-I mrna abundance in soleus and plantaris muscles decreased with spinal cord transection, indicating that it potentially serves a role in normal muscle to maintain muscle size. However, exercise and/or transplantation were not associated with an increase in IGF-I or IGF-II mrna abundance when measured 30 days after the intervention in either soleus or plantaris muscle compared with transection. Therefore, IGF-I mrna levels are not correlated with restoration of muscle mass. However, a role for IGFs in the decrease in atrophy and restoration of myonuclear number cannot be ruled out, as it is possible that a transient increase in IGF occurred shortly after initiation of the interventions. In summary, atrophy induced by spinal cord transection was reversed by the combination of exercise and transplantation implemented after a long postinjury interval in soleus and plantaris muscles. However, different cellular mechanisms appear to contribute to the restoration of mass in muscles with different fiber type compositions. Myofiber nuclei lost in soleus following spinal cord transection were restored with the combination of the interventions but not with either intervention alone. Satellite cell fusion is a potential mechanism by which these beneficial effects of the combined intervention are accomplished. Although IGFs do not appear to be involved in the restoration of muscle size or myofiber nuclear number, early responses to the interventions remain to be explored. On the other hand, plantaris muscles atrophied without significant loss of myofiber nuclear number and restoration of mass were not correlated with a change in myofiber nuclear number, suggesting that considerable variability exists in the response of different muscles to stimuli that alter muscle size. This research was supported by National Institutes of Health Grant HD E. E. Dupont-Versteegden was supported by a National Institute of Arthritis and Musculoskeletal and Skin Diseases National Research Service Award, and R. J. L. Murphy was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. Present address of R. J. L. Murphy: School of Recreation Management and Kinesiology, Acadia University, Wolfville, Nova Scotia BOP 1XO, Canada. REFERENCES 1. Adams GR. Role of insulin-like growth factor-i in the regulation of skeletal muscle adaptation to increased loading. Exerc Sport Sci Rev 26: 31 60, Adams GR and Haddad F. 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