Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise

Muscles ofspinal cord-transected rats exhibit severe atrophy and a shift toward afaster phenotype. Exercise can partially prevent these changes. Thegoal of this study was to investigate early events involved inregulating the muscle response to spinal transection and passivehindlimb exercise. Adult female Sprague-Dawley rats were anesthetized,and a complete spinal cord transection lesion(T₁₀) was created in all ratsexcept controls. Rats were killed 5 or 10 days after transection orthey were exercised daily on motor-driven bicycles starting at 5 daysafter transection and were killed 0.5, 1, or 5 days after the firstbout of exercise. Structural and biochemical features of soleus andextensor digitorum longus (EDL) muscles were studied. Atrophy wasdecreased in all fiber types of soleus and in type 2a and type 2xfibers of EDL after 5 days of exercise. However, exercise did notappear to affect fiber type that was altered within 5 days of spinalcord transection: fibers expressing myosin heavy chain 2xincreased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD waspreferentially expressed in satellite cell nuclei, and in EDL, MyoD wasmore readily detectable in myofiber nuclei, suggesting that MyoD andmyogenin have different functions in different muscles. Exercise didnot affect the level or localization of MyoD and myogenin expression.Similarly, Id-1 expression was transiently increased in soleus and EDLupon spinal cord transection, and no effect of exercise was observed.These results indicate that passive exercise can ameliorate muscleatrophy after spinal cord transection and that satellite cellactivation may play a role in muscle plasticity in response to spinalcord transection and exercise. Finally, the mechanisms underlyingmaintenance of muscle mass are likely distinct from those controllingmyosin heavy chain expression.

     

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

We have shown thatcycling exercise combined with fetal spinal cord transplantationrestored muscle mass reduced as a result of complete transection of thespinal cord. In this study, mechanisms whereby this combinedintervention increased the size of atrophied soleus and plantarismuscles were investigated. Rats were divided into five groups(n = 4, per group): control, nontransected; spinal cordtransected at T10 for 8 wk (Tx); spinal cord transected for 8 wk andexercised for the last 4 wk (TxEx); spinal cord transected for 8 wkwith 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 todeath (TxExTp). Tx soleus and plantaris muscles were decreased in sizecompared with control. Exercise and transplantation alone did notrestore muscle size in soleus, but exercise alone minimized atrophy inplantaris. However, the combination of exercise and transplantationresulted in a significant increase in muscle size in soleus andplantaris compared with transection alone. Furthermore, myofibernuclear number of soleus was decreased by 40% in Tx and was notaffected in TxEx or TxTp but was restored in TxExTp. A strongcorrelation (r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not inplantaris muscle, in which myonuclear number did not change with any ofthe experimental manipulations. 5'-Bromo-2'-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp 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 becontrolled by the insulin-like growth factors (IGF), as IGF-I andIGF-II mRNA abundance was decreased in Tx soleus and plantaris, and wasnot restored with the interventions. These results indicate that,following a relatively long postinjury interval, exercise andtransplantation combined restore muscle size. Satellite cell fusion andrestoration of myofiber nuclear number contributed to increased musclesize in the soleus, but not in plantaris, suggesting that cellularmechanisms regulating muscle size differ between muscles with differentfiber type composition.

     

A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy

Slow-twitch skeletal muscle atrophies greatly inresponse to unloading conditions. The cellular mechanisms thatcontribute to the restoration of muscle mass after atrophy are largelyunknown. Here, we show that atrophy of the mouse soleus is associatedwith a 36% decrease in myonuclear number after 2 wk of hindlimbsuspension. Myonuclear number is restored to control values during the2-wk recovery period in which muscle mass returns to normal, suggesting that muscle precursor cells proliferate and fuse with myofibers. Inhibition of muscle precursor cell proliferation by local-irradiation of the hindlimb completely prevents this increase inmyonuclear number. Muscle growth occurs normally during the first weekin irradiated muscles, but growth during the second week is inhibited, leading to a 50% attenuation in the restoration of muscle mass. Thusearly muscle growth occurs independently of an increase in myonuclearnumber, whereas later growth requires proliferating muscle precursorcells leading to myonuclear accretion. These results suggest thatincreasing the proliferative capacity of muscle precursor cells mayenhance restoration of muscle mass after atrophy.

     

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle.

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.     

Satellite cell regulation of muscle mass is altered at old age.

Muscle mass is decreased with advancing age, likely due to altered regulation of muscle fiber size. This study was designed to investigate cellular mechanisms contributing to this process. Analysis of male Fischer 344 X Brown Norway rats at 6, 20, and 32 mo of age demonstrated that, even though significant atrophy had occurred in soleus muscle by old age, myofiber nuclear number did not change, resulting in a decreased myonuclear domain. Also, the number of centrally located nuclei was significantly elevated in soleus muscle of 32-mo-old rats, correlating with an increase in gene expression of MyoD and myogenin. Whereas total 5'-bromo-2'deoxyuridine (BrdU)-positive nuclei were decreased at older ages, BrdU-positive myofiber nuclei were increased. These results suggest that, with age, loss of muscle mass is accompanied by increased myofiber nuclear density that involves fusion of proliferative satellite cells, resembling ongoing regeneration. Interestingly, centrally located myofiber nuclei were not BrdU labeled. Rats were subjected to hindlimb suspension (HS) for 7 or 14 days and intermittent reloading during HS for 1 h each day (IR) to investigate how aging affects the response of soleus muscle to disuse and an atrophy-reducing intervention. After 14 days of HS, soleus muscle size was decreased to a similar extent at all three ages. However, myofiber nuclear number and the total number of BrdU-positive nuclei decreased with HS only in the young rats. IR was associated with an attenuation of atrophy in soleus muscles of 6- and 20- but not 32-mo-old rats. Furthermore, IR was associated with an increase in BrdU-positive myofiber nuclei only in young rats. These data indicate that altered satellite cell function with age contributes to the impaired response of soleus muscle to an intervention that attenuates muscle atrophy in young animals during imposed disuse.     

No decrease in myonuclear number after long-term denervation in mature mice

Age-related but not artificially inducedmuscle fiber atrophy has been shown to occur without any decrease inmyonuclear number, although these results remain controversial. Thepresent study was carried out to clarify whether age difference affectsthe degree of decrease in myonuclear number occurring withdenervation-induced fiber atrophy. After denervation of 3-wk-old(young) and 4-mo-old (mature) mice, single myofibers were isolated fromthe plantaris muscles by alkali maceration, and their fibercross-sectional area (CSA), myonuclear number, andcytoplasm-to-myonucleus (C/N) ratios were analyzed. Fiber CSA in bothyoung and mature mice decreased with denervation. Myonuclear numberdecreased in young mice 5 and 10 days after denervation but wasunchanged in mature mice 10 and 120 days after denervation. C/N ratiodecreased in mature mice but was unchanged in denervated young mice.These results suggest that age differences affect the degree ofdecrease of myonuclear number with denervation and that fibercytoplasmic atrophy may occur without decrease in myonuclear number.

     

Aging influences cellular and molecular responses of apoptosis to skeletal muscle unloading

The influence of aging on skeletal myocyte apoptosis is not well understood. In this study we examined apoptosis and apoptotic regulatory factor responses to muscle atrophy induced via limb unloading following loading-induced hypertrophy. Muscle hypertrophy was induced by attaching a weight to one wing of young and aged Japanese quails for 14 days. Removing the weight for 7 or 14 days after the initial 14 days of loading induced muscle atrophy. The contralateral wing served as the intra-animal control. A time-released bromodeoxyuridine (BrdU) pellet was implanted subcutaneously with wing weighting to identify activated satellite cells/muscle precursor cells throughout the experimental period. Bcl-2 mRNA and protein levels decreased after 7 days of unloading, but they were unchanged after 14 days of unloading in young muscles. Bcl-2 protein level but not mRNA level decreased after 7 days of unloading in muscles of aged birds. Seven days of unloading increased the mRNA level of Bax in muscles from both young and aged birds. Fourteen days of unloading increased mRNA and protein levels of Bcl-2, decreased protein levels of Bax, and decreased nuclear apoptosis-inducing factor (AIF) protein level in muscles of aged birds. BrdU-positive nuclei were found in all unloaded muscles from both age groups, but the number of BrdU-positive nuclei relative to the total nuclei decreased after 14 days of unloading compared with 7 days of unloading. The TdT-mediated dUTP nick end labeling (TUNEL) index was higher after 7 days of unloading in both young and aged muscles and after 14 days of unloading in aged muscles. Immunofluorescent staining revealed that almost all of the TUNEL-positive nuclei were also BrdU immunopositive, suggesting that activated satellite cell nuclei (both fused and nonfused) underwent nuclear apoptosis during unloading. There were significant correlations among levels of Bcl-2, Bax, and AIF and TUNEL index. Our data are consistent with the hypothesis that apoptosis regulates, at least in part, unloading-induced muscle atrophy and loss of activated satellite cell nuclei in previously loaded muscles. Moreover, these data suggest that aging influences the apoptotic responses to prolonged unloading following hypertrophy in skeletal myocytes. satellite cells; Bcl-2 protein family     

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age‐related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein‐7 (Pax7) or laminin‐2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age‐related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre‐associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7⁺ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age‐dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.     

Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization

Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination.     

Cellular adaptation of the trapezius muscle in strength-trained athletes

 The aim of this study was to elucidate the cellular events that occur in the trapezius muscle following several years of strength training. In muscle biopsies from ten elite power lifters (PL) and six control subjects (C), several parameters were studied: cross-sectional area of muscle fibres, myosin heavy chain composition (MHC) and capillary supply [capillaries around fibres (CAF) and CAF/fibre area]. A method was also developed for counting the number of myonuclei and satellite cell nuclei. The proportion of fibres expressing MHC IIA, the cross-sectional area of each fibre type and the number of myonuclei, satellite cells and fibres expressing markers for early myogenesis were significantly higher in PL than in C (P<0.05). A significant correlation between the myonuclear number and the cross-sectional area was observed. Since myonuclei in mature muscle fibres are not able to divide, we suggest that the incorporation of satellite cell nuclei into muscle fibres resulted in the maintenance of a constant nuclear to cytoplasmic ratio. The presence of small diameter fibres expressing markers for early myogenesis indicates the formation of new muscle fibres. Accepted: 17 November 1998     

Establishment and assessment of a simple and easily reproducible incision model of spinal cord neuron cells in vitro

A growing number of in vitro models have been introduced to study the mechanisms of spinal cord injury. A potential drawback of these models is that they are difficult to reproduce. In this study, an in vitro incision model was established using primary cultured neuronal cells from fetal rat spinal cords. The neurons were subjected to incision in a simple and reproducible way. To assess whether this model could simulate the responses of spinal cord neuron cells in vivo after a spinal cord transection, apoptosis, and the expression of immediate early genes were detected in the neurons at various time points after injury. The results indicated that: (1) significantly more terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were observed at 1, 3, and 7 d following injury and (2) the expression of both c-Jun and c-Fos was induced 10 min after incision and had markedly higher levels 2 h post-injury. These results suggested that our model can partially imitate the responses of in vivo neuronal cells after a spinal cord transection and such models may facilitate further understanding of biochemical and cellular events associated with spinal cord injury.     

Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after spinal cord injury. I. Effects of total spinal cord transection

The contractile properties of motor units (MUs) were electrophysiologically investigated in the medial gastrocnemius (MG) muscle in 17 Wistar three-month-old female rats: 14, 30, 90 and 180 days after the total transection of the thoracic spinal cord and compared to those in intact (control) rats. A sag phenomenon, regularly observed in unfused tetani of fast units in intact animals at 40 Hz stimulation, almost completely disappeared in spinal rats. Therefore, the MUs of intact and spinal rats were classified as fast or slow types basing on 20 Hz tetanus index, the value of which was lower or equal 2.0 for fast and higher than 2.0 for slow MUs. The MUs composition of MG muscle changed with time after the spinal cord transection: an increasing proportion of fast fatigable (FF) units starting one month after injury and a disappearance of slow (S) units within the three months were observed. In all MUs investigated the twitch contraction and half-relaxation time were significantly prolonged after injury (p < 0.01, Mann–Whitney U-test). Moreover, a decrease of the fatigue index for fast resistant (FR) and slow MUs was observed in subsequent groups of spinal rats. No significant changes were found between twitch forces in all MU types of spinal animals (p > 0.05). However, due to a decrease of the maximal tetanic force, a significant rise of the twitch-to-tetanus ratio of all MUs in spinal rats was detected (p < 0.01). The considerable reduction of ability to potentiate the force was noticed for fast, especially FF type MUs. In conclusion, the spinal cord transection leads to changes in the proportion of the three MU types in rat MG muscle. The majority of changes in MUs’ contractile properties were developed progressively with time after the spinal cord injury. However, the most intensive alterations of twitch-time parameters were observed in rats one month after the transection.     

Role of muscle progenitor cells in maintaining morphological characteristics of rat soleus muscle during gravitational unloading by means of passive stretch

Working hypertrophy of skeletal muscle is usually coupled with activation of satellite cells with subsequent incorporation of their nuclei into muscle fibers. Earlier, it has been repeatedly shown that muscle stretching prevents the development of atrophic alterations and is accompanied by an intensification of protein synthesis. We suggested that the elimination of the proliferative abilities of progenitor cells by γ-irradiation would lead to a partial loss of the ability of muscle fibers to maintain their size. To evaluate the role of progenitor cells in the development of the preventive effect of passive stretching, an experiment was carried out with the 2500 rad local irradiation of a rat shin and subsequent hind-limb suspension or hind-limb suspension with stretch. Passive stretching during hind-limb suspension completely prevented atrophy, the transformation of fibers, and a decrease in the myonuclear number observed in the hind-limb-suspension group. Irradiation produced no action of the preventive effect of passive stretch. The conclusion is made that passive stretch preventive action is also realized in the absence of proliferating satellite cells.     

Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training

A skeletal muscle fibre maintains its cytoplasmic volume by means of hundreds of myonuclei distributed along its entire length. Therefore it is hypothesised that changes in fibre size would involve modifications in myonuclear number. In this study, we have examined whether 10 weeks of strength training can induce changes in the number of myonuclei and satellite cells in female trapezius muscles. Biopsies were taken pre- and posttraining from the upper part of the descending trapezius muscle of nine subjects. Muscle samples were analysed for fibre area and myonuclear and satellite cell number using immunohistochemistry. There was a 36% increase in the cross-sectional area of muscle fibres. The hypertrophy of muscle fibres was accompanied by an approximately 70% increase in myonuclear number and a 46% increase in the number of satellite cells. Myonuclei number was positively correlated to satellite cell number indicating that a muscle with an increased concentration of myonuclei will contain a correspondingly higher number of satellite cells. The acquisition of additional myonuclei appears to be required to support the enlargement of multinucleated muscle cells following 10 weeks of strength training. Increased satellite cell content suggests that mitotic divisions of satellite cells produced daughter cells that became satellite cells. Accepted: 30 November 1999     

The COX-2 pathway regulates growth of atrophied muscle via multiple mechanisms

Loss of muscle mass occurs with disease, injury, aging, and inactivity. Restoration of normal muscle mass depends on myofiber growth, the regulation of which is incompletely understood. Cyclooxygenase (COX)-2 is one of two isoforms of COX that catalyzes the synthesis of prostaglandins, paracrine hormones that regulate diverse physiological and pathophysiological processes. Previously, we demonstrated that the COX-2 pathway regulates early stages of myofiber growth during muscle regeneration. However, whether the COX-2 pathway plays a common role in adult myofiber growth or functions specifically during muscle regeneration is unknown. Therefore, we examined the role of COX-2 during myofiber growth following atrophy in mice. Muscle atrophy was induced by hindlimb suspension (HS) for 2 wk, followed by a reloading period, during which mice were treated with either the COX-2-selective inhibitor SC-236 (6 mg·kg^–1·day^–1) or vehicle. COX-2 protein was expressed and SC-236 attenuated myofiber growth during reloading in both soleus and plantaris muscles. Attenuated myofiber growth in the soleus was associated with both decreased myonuclear addition and decreased inflammation, whereas neither of these processes mediated the effects of SC-236 on plantaris growth. In addition, COX-2^–/– satellite cells exhibited impaired activation/proliferation in vitro, suggesting direct regulation of muscle cell activity by COX-2. Together, these data suggest that the COX-2 pathway plays a common regulatory role during various types of muscle growth via multiple mechanisms. cyclooxygenase-2; prostaglandins; myonuclear number; satellite cells; inflammation     

Optimal Location and Time for Neural Stem Cell Transplantation into Transected Rat Spinal Cord

Implanted neural stem cells (NSC) could improve neurological functions following spinal cord injury (SCI), but the optimal conditions for NSC transplantation are largely unknown, especially in transected spinal cord. This study investigated the effect and fate of NSC engrafted into spinal cords at different locations and time points following T₉ spinal cord transection. Engrafted NSC could survive and migrate in host spinal cords. Significant improvement in hindlimb locomotor functions associated with NSC survival was found in rats receiving NSC transplantation in the spinal cords rostral to the transection site at the subacute stage (7 days post operation), compared with those caudal to the transection site at the acute stage (at the time of injury). At 4 weeks post operation, CD68 immunohistochemical staining confirmed that macrophages were less in rostrally transplanted sites and in subacute groups than seen in caudal and acute transplanted rats. The present findings indicated that NSC transplantation into spinal cords rostral to transection site at the subacute stage is an optimal strategy for engrafted NSC survival and host behavioral improvement. It therefore would be available to the usage of NSC for the treatment of SCI in the future clinic trial.     

Mechanical load-dependent regulation of satellite cell and fiber size in rat soleus muscle

The effects of mechanical unloading and reloading on the properties of rat soleus muscle fibers were investigated in male Wistar Hannover rats. Satellite cells in the fibers of control rats were distributed evenly throughout the fiber length. After 16 days of hindlimb unloading, the number of satellite cells in the central, but not the proximal or distal, region of the fiber was decreased. The number of satellite cells in the central region gradually increased during the 16-day period of reloading. The mean sarcomere length in the central region of the fibers was passively shortened during unloading due to the plantarflexed position at the ankle joint: sarcomere length was maintained at <2.1 µm, which is a critical length for tension development. Myonuclear number and domain size, fiber cross-sectional area, and the total number of mitotically active and quiescent satellite cells of whole muscle fibers were lower than control fibers after 16 days of unloading. These values then returned to control values after 16 days of reloading. These results suggest that satellite cells play an important role in the regulation of muscle fiber properties. The data also indicate that the satellite cell-related regulation of muscle fiber properties is dependent on the level of mechanical loading, which, in turn, is influenced by the mean sarcomere length. However, it is still unclear why the region-specific responses, which were obvious in satellite cells, were not induced in myonuclear number and fiber cross-sectional area. sarcomere     

Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype.

A number of significant advances have been developed for treating spinal cord injury during the past two decades. The combination of peripheral nerve grafts and acidic fibroblast growth factor (hereafter referred to as PNG) has been shown to partially restore hindlimb function. However, very little is known about the effects of such treatments in restoring normal muscle phenotype. The primary goal of the current study was to test the hypothesis that PNG would completely or partially restore 1) muscle mass and muscle fiber cross-sectional area and 2) the slow myosin heavy chain phenotype of the soleus muscle. To test this hypothesis, we assigned female Sprague-Dawley rats to three groups: 1) sham control, 2) spinal cord transection (Tx), and 3) spinal cord transection plus PNG (Tx+PNG). Six months following spinal cord transection, the open-field test was performed to assess locomotor function, and then the soleus muscles were harvested and analyzed. SDS-PAGE for single muscle fiber was used to evaluate the myosin heavy chain (MHC) isoform expression pattern following the injury and treatment. Immunohistochemistry was used to identify serotonin (5-HT) fibers in the spinal cord. Compared with the Tx group, the Tx+PNG group showed 1) significantly improved Basso, Beattie, and Bresnahan scores (hindlimb locomotion test), 2) less muscle atrophy, 3) a higher percentage of slow type I fibers, and 4) 5-HT fibers distal to the lesion site. We conclude that the combined treatment of PNG is partially effective in restoring the muscle mass and slow phenotype of the soleus muscle in a T-8 spinal cord-transected rat model.     

Structural and functional regeneration after spinal cord injury in the weakly electric teleost fish, <Emphasis Type="Italic">Apteronotus</Emphasis><Emphasis Type="Italic">leptorhynchus</Emphasis>

In contrast to mammals, teleost fish exhibit an enormous potential to regenerate adult spinal cord tissue after injury. However, the mechanisms mediating this ability are largely unknown. Here, we analyzed the major processes underlying structural and functional regeneration after amputation of the caudal portion of the spinal cord in Apteronotus leptorhynchus, a weakly electric teleost. After a transient wave of apoptotic cell death, cell proliferation started to increase 5 days after the lesion and persisted at high levels for at least 50 days. New cells differentiated into neurons, glia, and ependymal cells. Retrograde tract tracing revealed axonal re-growth and innervation of the regenerate. Functional regeneration was demonstrated by recovery of the amplitude of the electric organ discharge, a behavior generated by spinal motoneurons. Computer simulations indicated that the observed rates of apoptotic cell death and cell proliferation can adequately explain the re-growth of the spinal cord. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.