Histochem Cell Biol (2006) 126: 409417 DOI 10.

1007/s00418-006-0176-3

OR I G I NA L P AP E R

Anders Eriksson Mona Lindstrm Lena Carlsson Lars-Eric Thornell

Hypertrophic muscle fibers with fissures in power-lifters; fiber splitting or defect regeneration?

Accepted: 8 March 2006 / Published online: 20 April 2006 Springer-Verlag 2006

Abstract Power-lifters have hypertrophic muscle Wbers with Wssures seen in cross-sections, called as Wber splitting.Whether this phenomenon is due to real splitting or defective regeneration has not been settled. To elucidate this matter,we have examined biopsies from the trapezius and vastus lateralis of power lifters (P group) and power lifters self-administrating anabolic steroids (PAS group). For this purpose, immunohistochemical staining of serial cross -sections was used. The PAS group had signiWcantly more Wbers with Wssures than the P group in the vastus lateralis (1.2%0.95% vs 0.350.34, P<0.05) but not in the trapezius muscle (1.7% in both groups). Serial sections revealed that the Wbers with Wssures changed their proWle profoundly over short distances. Some such Wbers had a mature staining proWle, whereas other Wbers indicated recent degeneration and/or regeneration. Activation of satellite cells and formation of aberrant segments were also evident. We conclude that the so-called split Wbers are due to defect regeneration. Some Wbers with Wssures are the results of old events of segmental muscle Wber damage, whereas the others reXect an ongoing process. The normal regenerative process is most likely disturbed in power-lifters by their continuous training with repeated high mechanical stress on the muscles. Keywords Split Wber Anabolic steroids Abortive regeneration

Introduction
The term muscle-Wber splitting is widely used and refers to muscle Wbers that seem to be divided or fragmented into two or more smaller, daughter Wbers (Antonio and Gonyea 1993; Gonyea et al. 1977; Swash and Schwartz 1977). This feature is most common in diVerent myopathies such as Duchennes muscle dystrophy (DMD), polymyositis and dermatomyositis (Richards et al. 1988; Schwartz et al. 1976), These observations lead some authors in early studies to consider muscle Wber splitting as a degenerative change (Dubowitz et al. 1973; Kihira and Nonaka 1985). Another prevalent theory of the 1970s was that when a Wber reaches a critical size, the supply of oxygen and exchange of metabolites are no longer eYcient and the Wber splits to reduce the diVusion distance (Swash and Schwartz 1977). Evidences in favor of this theory are the obvious split appearance and the separated parts showing the same histochemical characteristics. In power athletes split Wbers are commonly seen, leading some authors to consider muscle Wber splitting as a source of hyperplasia, both in weight-lifting humans (Larsson and Tesch 1986; Tesch 1988) and weight-lifting animals (Gallanti et al. 1992; Gonyea et al. 1977; Gonyea 1980; Ho et al. 1980). Schmalbruch on the other hand maintained that split Wbers were due to the replacement of necrotic Wbers from activation of satellite cells (Schmalbruch 1976), whereas others suggested that they could be due to the activation of satellite cells even in the absence of muscle Wber necrosis (James 1973; Danon and Carpenter 1991). Also, diVerent types of split Wbers have been reported (Swash and Schwartz 1977). In neuropathies, Swash and Schwartz assumed mechanical overload induced splitting of Wbers that were still innervated whereas in polymyositis, regeneration following segmental necrosis was a main factor, inducing split muscle Wbers (Swash and Schwartz 1977). It has also been suggested that split Wbers can be related to the eVects of denervation and reinnervation (Balaram et al. 1997; Chou and Nonaka 1977).

A. Eriksson M. Lindstrm L. Carlsson L. E. Thornell (&) Department of Integrative Medical Biology, Section for Anatomy, Umea University, 901 87 Umea, Sweden E-mail: lars-eric.thornell@anatomy.umu.se Tel.: +46-90-7865142 Fax: +46-90-7865480 A. Eriksson Department of Health Science, Section for Medical Science, Lulea University of Technology, 961 36 Boden, Sweden

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The mechanism for how muscle Wbers become divided or fragmented is still not settled. In the recent textbook Myology (Engel and Franzini-Armstrong 2004) Wbers with a split appearance are considered to be the eVect of splitting or branching, whereas in others, like Pathology of skeletal muscles (Carpenter and Karpati 2001), all the above alternative hypotheses are discussed. In our previous studies on power-lifters and powerlifters using anabolic steroids, we observed a massive muscle Wber hypertrophy and muscle Wber morphology, indicating split Wbers (Eriksson et al. 2005; Kadi et al. 1999b). The aim of the present study was to elucidate the mechanisms behind muscle Wber splitting in power-lifters. Serial cross sections in biopsies from both the vastus lateralis and the trapezius muscles of high-level power-lifters were examined. Two groups of power-lifters were compared; one group who had used anabolic steroids for several years (PAS) and another group who had never used these substances (P). Healthy nonpower-lifters served as controls. The major Wnding in the present study was that defect regeneration is the mechanism behind the branched muscle Wbers in power lifters.

Enzymehistochemistry and routine histology Serial, 610 m thick cross-sections were cut at 20C using a Reichert Jung cryostat (Leica, Nussloch, Germany), then mounted on glass slides and air-dried at room temperature. The sections were stained for the demonstration of myoWbrillar ATPase after alkaline (pH 10.4 and 9.4) and acid (pH 4.6 and 4.3) pre-incubations (Dubowitz 1985). Other sections were stained with hematoxylin-eosin (htx-eosin), nicotinamide adenin dinucleotide tetrazolium reductase (NADH-TR) and Gomori trichrome (GT) (Dubowitz 1985). Immunohistochemistry Five m thick cross-sections, serial to those used for enzyme histochemistry, were used for immunohistochemical analysis. Sections were rehydrated in 0.01 M PBS, immersed in 5% non-immune serum and incubated with primary antibodies for 60 min at +37C or overnight at +4C. Visualization of bound antibodies was performed with peroxidaseanti-peroxidase (PAP) technique (Sternberger 1979) using antibodies from Dako (Glostrup, Denmark), or with indirect immunoXuorescence, using Xuorescein (FITC) and rhodamine red-X for green and red Xuorescence, respectively (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Double labeling was performed with one monoclonal and one polyclonal antibody, visualized by secondary antibodies coupled to Xuorochromes of diVerent wavelengths. Nuclei were identiWed with 4,6-diamino-2phenylindole (DAPI). Control sections were treated according to standard protocols except that the primary antibody was exchanged with non-immune serum. The sections were evaluated in a Nikon eclipse E 800 microscope (Nikon Inc., Melville, NY, USA) and a SPOT RT Color camera (Dignostic Instruments Inc., Sterling Heights, MI, USA) was used for image acquisition. Digital images were processed using the Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA, USA). Antibodies Myosin heavy chain (MyHC) composition was assessed using well-characterized monoclonal antibodies (mAbs); A4.840 strongly stains type I Wbers and moderately stains IIC Wbers, N2.261 strongly stains type IIA and IIC Wbers, weakly stains type I and IIAB Wbers, whereas type IIB Wbers were unstained. F1.652 and NCL-MHCn detect embryonic and fetal MyHC isoforms, respectively. The MyHC Abs were purchased from Developmental Studies Hybridoma Bank, Iowa and Novocastra Laboratories Ltd. UK. MAb NCL-Merosin against laminin 2 chain (Aumailley et al. 2005) (Novocastra Laboratories Ltd. UK), mAb 4C7 against laminin 5-chain (provided by I Virtanen, Helsinki, Finland) and polyclonal Ab PC128 against human laminin (The Binding Site Ltd, Birming-

Materials and methods

Nineteen power-lifters participated in the study. Nine of the subjects (31.43.3 years) have reported the use of a wide variety of high doses of testosterone and anabolic steroids for a period of 93.3 years, whereas ten other power-lifters (27.77.5 years) have never used these substances. All athletes were highly competitive and participated regularly in national and/or international competitions in power events. They trained regularly for four to six times a week, two to three hours per session. The sessions consisted of four to seven sets of exercises and three to twelve repetitions per set. As controls, vastus lateralis and trapezius biopsies from Wve sedentary people were used. All subjects gave their informed consent to participate in the present study. The Ethical Committee of Umea University approved this work. Written consent in accordance with the World Medical Associations Declaration of Helsinki was obtained from all the subjects. Muscle samples Biopsies were taken from the descending part of the trapezius muscle and the upper ventral part of the vastus lateralis muscle. No exercise session was performed on the day of the biopsy. The samples were mounted on Tissue Tek OCT Compound (Tissue Tek, Miles laboratories, Elkhart, USA) and quickly frozen in propane, cooled in liquid nitrogen and stored at 80C until analyzed.

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ham, UK) were used for identiWcation of the basement membrane of muscle Wbers. MAb NCL-Dys2 against dystrophin was used for detection of the plasma membrane (Novocastra Laboratories Ltd., UK) and the intermediate Wlament proteins desmin and vimentin were identiWed with the mAbs D33 and V9 (Dako, Glostrup, Denmark). Sections were also labeled with the mAb CD56 (Becton Dickinson Immunocytochemistry Systems, San Jose, CA, USA) against the Leu 19 antigen, which is expressed during early stages of muscle Wber formation and in satellite cells (Kadi et al. 1999a). Light microscopy and morphometric analysis The proportion of muscle Wbers with Wssures and Wbers that occupied an area equivalent to that of one Wber was photographed and counted on whole muscle cross sections (mean 449 Wbers) using a microscope (Zeiss Axiophot, Carl Zeiss, Oberkochen, Germany). The proportion of branched Wbers was calculated as follows; (number of split Wbers)/(total number of myoWbers)100. On sections stained for htx-eosin, two randomly chosen areas from each biopsy were scanned to determine the frequency of Wbers containing internal myonuclei. Statistical analysis Data are presented as means and standard deviations. Because data were normally distributed, the statistical signiWcance of the diVerences between the two groups was determined using a t-test for unpaired data. The statistical signiWcance of correlations between two parameters was determined by using Fichers r to z test. P<0.05 were considered statistically signiWcant.

The control subjects lacked branched Wbers except for one subject who had a few branched Wbers in the trapezius muscle. Serial cross-sections In serial sections, the proWle of the muscle Wbers changed along their length (Fig. 1). Fissures varied in depth, some partly and some completely dividing the Wbers, whereas others disappeared i.e. the branched parts merged into one Wber. Some Wssures could be followed over a distance up to 200 m (Fig. 1). In one Wber where the branched parts merged, a new branch appeared again after 90 m. Branched Wbers were in general of the same Wber type and showed the same staining pattern in both enzymehistochemical stainings for myoWbrillar ATP-ases and immunostainings against diVerent MyHC isoforms. However two exceptions were seen; one small part of a branched Wber separated by a basement membrane (not shown) stained positively for Leu 19, (Fig. 3A), whereas in another Wber, the small part stained weaker than the large part with the antibody N2.261 for MyHCs (Fig. 3B). Interestingly, the Wber in Fig. 3A did not stain diVerently with respect to MyHC (not shown) and the Wber in Fig. 3B did not stain for Leu 19 (not shown). Myonuclei In both groups, internal myonuclei was a prominent feature both in the vastus lateralis and the trapezius muscles (Eriksson et al. 2005; Kadi 2000; Kadi et al. 1999b). In Wbers with Wssures, there were often internal nuclei placed at the central part of the Wssure. In serial sections, the Wssures were sometimes preceded by myonuclei (Fig. 1 D3). The number of Wbers with internal myonuclei correlated signiWcantly with the proportion of branched Wbers in the vastus lateralis muscle (r=0,53, P<0.05) when data for the PAS and the P groups were pooled. This was not seen in the trapezius muscle. Degeneration and regeneration In htx-eosin stained sections, a few pale Wbers were seen. These Wbers were distinguishable also in NADH-TR and GT (not shown) and were considered necrotic. In serial cross sections, these Wbers lacked immunostaining with antibodies against dystrophin (Fig. 4A) but showed preserved staining for laminin (Fig. 4B). Myocytes, detected by their staining for embryonic and fetal (not shown) MyHCs were present inside the basement membrane (Fig. 4C). They also stained positive for vimentin (Fig. 4D), desmin (Fig. 4E) and Leu 19 (Fig. 4F), a staining proWle indicative of an ongoing regeneration process. An irregular staining of pale Wbers in htx-eosin stained sections, suggests the presence of debris and invading cells in these Wbers (not shown). These non-muscle cells were visualized by staining for vimentin (Fig. 4D). Some Wbers with basophilic cytoplasm and vesicular nuclei were also seen (Fig. 5A). These W bers could

Results
Branched muscle Wbers were primarily identiWed in cross sections stained with htx-eosin. Muscle Wbers with Wssures and compound Wbers that occupied an area equivalent to that of one Wber, but that were composed of two or more closely apposed Wbers were considered as one branched Wber (Fig. 1). In the vastus lateralis muscle, branched muscle Wbers were seen in all PAS subjects and in eight out of ten P subjects. The mean proportion of branched Wbers diVered signiWcantly between the groups (PAS; 1.2%0.95% (mean SD) and P; 0.35% 0.34%, P<0.05) (Table 1). In the trapezius muscle, branched muscle Wbers were observed in eight out of nine PAS subjects and in six out of ten P subjects. The proportion of branched Wbers was of the same magnitude in the two groups (PAS; 1.7% 1.8% and P; 1.7% 2.8%, P>0.9) (Table 1). In the PAS group, a signiWcant correlation between the proportion of branched Wbers and Wber area in the trapezius muscle was seen (r=0.67, P<0.05) (Fig. 2).

412 Fig. 1 Serial cross-sections of a trapezius biopsy from a PAS subject. Sections stained for htx eosin (A, B), NADH (C) and Gomori trichrome (D). A is the Wrst section in the series, B the second, C the fourth and D the seventh. The distance between the Wrst and the seventh section is approximately 50 m (A): Overview. Note branched Wbers with Wssures (arrows). Fibers marked with 1 to 4 are shown in BD. Several internal myonuclei are seen (arrowheads). B1D1 Note how the Wber 1 and Wber 1* merge and that the Wssure seen in A gradually disappears. B2 D2 A large Wber with a Wssure (arrow) and a small segment (*) is shown. The Wssure becomes deeper and Wnally three segments are seen. B3D3 Fiber 3a, the Wssure between 3a and 3* gradually disappears whereas in 3b the Wssure gradually separates the Wber into two branches. Internal myonuclei (arrowheads) mark the location of the Wssure in D1, D3. B4D4 The branch 4* separated by a cleft in A gradually melts together with Wber 4. Bars: AD = 25 m

also be distinguished from the surrounding normal Wbers in diVerent immunostainings (Fig. 5B-F). They showed prominent staining for the laminin 5 chain (Fig. 5C) stained for embryonic (Fig. 5D) and fetal MyHCs and Leu 19 (Fig. 5E), stainings normally absent in mature Wbers. They also had an increased staining for desmin (Fig. 5F). One of the basophilic

Wbers appeared as branched in the section stained for htx-eosin (Fig. 6A) but had a common basement membrane that stained with the antibodies against laminin ( 2 and 5 chains) (Fig. 6B and 6C). The Wber also showed strong staining for embryonic (Fig. 6D) and fetal MyHCs (not shown), Leu19 (not shown) and desmin (not shown).

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Discussion
In the present study, we have veriWed that muscle Wbers with Wssures that partly or totally divide a Wber are related to proWles of so called split Wbers and that this phenomenon is due to Wber branching. Several features of branched Wbers suggest that the branching is due to defect regeneration. Segmental muscle Wber disruption and necrosis were present in the power-lifters. The branched parts of the Wbers were often short segments and muscle Wber morphology considered typical, for regenerative muscle Wbers observed. One has to bear in mind that a biopsy only displays a glimpse of the history of the muscle. Several of the branched Wbers had a mature appearance, indicative of events that could have occurred a long time ago, whereas other Wber features indicate ongoing processes of degeneration and regeneration. All power lifters in this study had a training history of several years with 46 sessions a week, with loads of above 300 kg per repetition. Such muscle loads induce a high mechanical stress that can induce focal damage. Similarly, the largest and fastest Wbers in DMD are the Wrst to be aVected by mechanical stress, although in DMD the reason for the focal damage is the lack of dystrophin (Webster et al. 1988). Undoubtedly, segmental muscle Wber necrosis was present in the power lifters. Pale Wbers in htx-eosin lacked immunostaining with

Fig. 2 Scattergram of proportion of split Wbers versus Wber area in the trapezius muscle of the PAS group. The two parameters show a signiWcant correlation (r=0.67,P<0.05)

Single Wbre or clusters of very small sized Wbers with a variable staining pattern were observed, especially in the PAS group (Eriksson et al. 2005). All stained positive for laminin 5 chain and Leu 19 and had an increased staining for desmin, whereas the degree of staining for fetal MyHC varied (positive to negative).
Table 1 Individual and group data for the PAS and P groups Vastus lateralis Subject PAS 1 2 3 4 5 6 7 8 9 MeanSD 1 2 3 4 5 6 7 8 9 10 MeanSD Proportion of Wbers with internal nuclei 57.7 19.2 15.2 8.3 6.3 37.0 41.5 42.9 36.1 29.317.7a 19.2 20.8 3.8 2.3 5.0 29.5 0.0 2.0 2.6 8.1 9.310.1

Trapezius Proportion of split Wbers 2.0 2.1 0.6 0.3 0.9 0.8 3.0 0.4 0.6 1.20.95a 0.8 0.3 0.1 0.3 0.0 0.2 0.0 0.9 0.8 0.35 0.40.3 Proportion of Wbers with internal nuclei 32.0 22.0 27.0 5.0 45.0 18.0 11.0 37.0 27.0 24.912.5a 4.0 6.0 6.0 4.0 9.0 6.0 2.0 10.0 2.0 2.0 5.12.8 Proportion of split Wbers 3.4 0.4 5.2 1.3 1.3 0.0 0.5 2.0 0.2 1.71.8 2.0 0.7 8.8 0.1 0.0 3.3 0.0 2.2 0.0 0.0 1.72.8

Means signiWcant diVerence between the PAS and the P group for each muscle at P<0.05

414 Fig. 3 Biopsy of vastus lateralis muscle from a PAS subject. Sections stained for Leu 19 (A) and for mAb. N2.261 (MyHC). A A small muscle Wber stained for Leu 19 is separated by a narrow cleft from a larger Wber. B A small rounded Wber diVers in degree of staining for mAb. N2.261 as compared to the neighboring Wber. Both small Wbers were separated from the neighboring Wber with laminin (not shown). Bar 25 m

antibodies against dystrophin, which means that the plasma membrane was absent, and the persistent staining for laminin means that the basement membrane was preserved. Furthermore, staining for the intermediate Wlament protein desmin was lacking. All these features are typical for necrotic Wbers (Thornell et al. 1984).
Fig. 4 Serial cross-sections of a trapezius biopsy from a PAS subject. Sections stained for A dystrophin, B laminin, C embryonic MyHC (red) and laminin 5 chain (green), D vimentin, E desmin, and F Leu 19. All sections were counterstained with Dapi which stains nuclei blue. Note a necrotic Wber * which lacks staining for dystrophin but stains for laminin and which contains a number of nuclei related to non-muscle cells. Signs of regeneration are apparent such as staining for embryonic MyHCs, desmin and Leu 19. Note also the small Wber in A and B (arrows) that changes its appearance through the series. The Wber becomes several in CE and three in F. These Wbers also stain for laminin 5 chain, desmin and Leu 19 but not for embryonic MyHC and vimentin. Bar 25 m

Muscle Wber damage normally leads to regenerative attempts. Typical features of Wbers undergoing regeneration are basophilic cytoplasm and vesicular nuclei (Dubowitz 1974). This was observed in some Wbers in all power-lifters but not in the controls. Other features of regeneration such as satellite cell activation, evidenced

415 Fig. 5 Selected sections of a serie of 52 sections from a PAS subject trapezius biopsy. Sections stained for A htx-eosin, B laminin 2 chain, C laminin 5 chain, D embryonic MyHC, E Leu-19 and F desmin. Note that the Wber marked with an * diVers in staining pattern from neighboring Wbers throughout the series. Note how the Wber marked with an arrow changes appearance from two Wbers to three Wbers and four Wbers to Wnally end up as one single Wber. In E a satellite cell is marked with an arrowhead and shown in higher magniWcation in the inset. Bar 25 m

by the presence of myocyte and myotube formation within the laminin sheets of necrotic Wbers was also observed. In line with our previous Wndings (Thornell et al. 1980) such regenerating Wbers stained positive for embryonic and fetal MyHCs and Leu 19 and had an increased staining for desmin. They also had a laminin isoform composition typical of developmental or regenerating Wbers. Laminin 5 is a laminin isoform expressed early in developmental muscle Wbers as well as around

Wbers undergoing regeneration (Gullberg et al. 1999). The two small Wbers, which showed diVerent staining than their neighboring larger Wbers (Fig2), never merged with the larger Wbers and are thus interpreted to be independent Wbers formed by activated satellite cells. We further propose that the segmentally injured muscle Wbers in certain circumstances, will not heal properly. The myotubes may not be able to fuse within a damaged Wber when they are exposed to repeated stress.

Fig. 6 Serial cross-sections of a trapezius biopsy from a the PAS subject. Sections stained for A htx-eosin, B laminin 2 chain, C laminin 5 chain and D embryonic MyHC. In A the Wber seems divided into two separate parts whereas B and C show that the Wber is sur-

rounded by a common basal lamina. Note that the Wber stains stronger for laminin 5 chain than the surrounding Wbers. The Wber also stained strongly for embryonic MyHC (D). Bar 25 m

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The high incidence of internal nuclei in the power-lifters might also be a feature of regeneration. The proportion of branched Wbers in the vastus lateralis muscle but not in the trapezius muscle, correlated signiWcantly with the proportion of Wbers with internal myonuclei when the PAS and the P groups were taken together. So far, the cause of internal nuclei is debated (Schmalbruch 1999). One view is that the internal nuclei are the consequence of many years of degeneration and regeneration whereby nuclei have been trapped within the Wbers after fusion of regenerating myocytes and myotubes (Schmalbruch 1985). Data from tissue cultures have suggested that nuclei in myotubes are mobile (Englander and Rubin 1987), whereas it has been demonstrated that the nuclei have a Wxed position in mice muscle Wbers in vivo (Bruusgaard et al. 2003). Branched muscle Wbers were a typical feature of almost all power-lifters and the highest proportion was seen in the PAS subjects with the largest Wber areas. This supports the earlier studies suggesting that heavy resistance training in humans induce branched muscle Wbers (Larsson and Tesch 1986; Tesch 1988) and that branched Wbers are related to Wber size (Chou and Nonaka 1977; Edgerton 1970; Hall-Craggs 1970; Hall-Craggs and Lawrence 1970; Swash and Schwartz 1977). We propose that there are muscle dependent discrepancies in adaptations to high resistance training with and without anabolic steroids. We consider each human skeletal muscle or muscle group unique regarding its characteristics and its adaptation capacity. Here we show that power-lifters who self administrate anabolic steroids have a higher proportion of branched Wbers in the vastus lateralis but not in the trapezius muscle, compared to power-lifters who did not use these substances. In the trapezius muscle, the proportion of branched Wbers and the mean Wber area correlated in power-lifters who selfadministrate anabolic steroids. Athletes administrating anabolic steroids have been reported to have a higher training intensity and total work load compared to athletes who do not use these substances (Alen et al. 1984; Freed et al. 1975; Johnson and OShea 1969). This may imply higher mechanical stress per area unit in each contraction in these subjects. DiVerent contraction patterns in the vastus lateralis and the trapezius muscles in power-lifters might also induce diVerent cellular responses. The vastus lateralis is of importance preferentially in the squat event, when the lifter sits or squats down to a required depth. This lift takes approximately 25 s. In dead lift (lifting a barbell from the ground level) the trapezius muscle performs an isometric contraction for a total time of approximately 6 10 s. It is likely that these diVerences in utilization between the vastus lateralis and trapezius muscles might be reXected at the muscle Wber level (Eriksson et al. 2005). In conclusion, we propose that defect regeneration is the mechanism responsible for branched muscle Wbers in power-lifters and that internalization of myonuclei is an outcome of this process. We suggest that the term muscle Wber splitting can still be used in the context

of muscle morphology and pathology, as long as one keeps in mind that the term does not reXect its pathogenesis.
Acknowledgments We thank Margaretha Enerstedt and AnnaKarin Olofsson for excellent technical assistance, Fatima Pedrosa Domellf and Christer Malm for valuable comments on the manuscript and Fawzi Kadi for providing data on the number of internal nuclei in the trapezius. This study was supported by grants from the Swedish National Centre for research in sports CIF (109/05), the Swedish Research Council (12X-3934) and the Medical Faculty of Ume University.

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