The principal basis for estimation of muscle changes and inflammation in the rabbit soleus and gastrocnemius muscle after unilateral EMS/E.

<p>The parameters used are variability in fiber size (overall existence of small and large sized fibers), fibrosis (abnormal presence of connective tissue), frequency of internal nuclei (percentage of fibers containing internal nuclei), frequency of fiber splitting per unit area (split fibers per mm² muscle cross-sectional area), frequency of necrotic fibers per unit area (necrotic fibers per mm² muscle cross-sectional area) and inflammation (degree of inflammatory cell infiltration in the muscle).</p

Muscle fiber regeneration and degeneration.

<p>Serial sections from a exercised soleus muscle after 1 w of EMS/E. The sections are stained with H&E (A, D) and for embryonic MyHC (B), fetal MyHC (C), desmin (E) and fibronectin (F). Sections B and C are double stained for Laminin α-2 chain for visualization of the basement membrane of muscle fibers. Figures (A–C) show regenerating fibers (arrows) and a necrotic fiber (asterisks) (A, B). This necrotic fiber is shown in figures (D–F). Note the infiltration of inflammatory cells in the necrotic fiber (D), the lack of reactivity for desmin (E) and the extensive reactivity for fibronectin (F) in this fiber (Bar = 25 µm).</p

Histological changes in the soleus muscle after 1 w.

<p>Muscle samples from the exercised side (left column, A, C, E) and contrateral non-exercised side (right column, B, D, F) of the soleus muscle after 1 w of EMS/E. The sections are stained with H&E. The left column (exercised side) shows fiber hypertrophy and fiber splitting (arrow) (A), small angular fibers (arrowheads) (C) and existence of internal nuclei (arrow) (C) and inflammatory cell infiltration in the area of a necrotic fiber (asterisks) (E). The right column (non-exercised side) shows occurrence of fiber splitting (arrow) (B), internal nuclei (arrow), fiber hypertrophy (D) and an accumulation of inflammatory cells in the extracellular matrix (arrow) (F). (Bar = 50 µm).</p

Serial sections of nerve fascicles.

<p>Cross-sections of a nerve fascicle from soleus muscle (non-exercised side) after 6w of unilateral EMS/E. The sections are stained with H&E (A), mAb S-100beta (B) and mAb S-100beta and DAPI (C). Note the marked presence of connective tissue and a high number of cell nuclei within the nerve fascicle (A). In (B), mAb S-100beta stains Schwann cells. Figure (C) shows two patterns of stained nuclei, one bluish and one pink, where the bluish nuclei are stained only for DAPI. Some of the nuclei located in cell cytoplasm are devoid of S-100beta reaction (small arrows) whilst others nuclei in the cytoplasm exhibits a S-100beta reaction around the nuclei (arrowheads). The cells with pink nuclei showed immunoreaction in the cytoplasm for mAb S-100beta (large arrows). (Original magnification×200).</p

Mean values of the scoring of variability in fiber size, fibrosis, percentage of fiber with increased internal nuclei, fiber splitting per unit area, presence of necrotic fibers per unit area and degree of inflammatory cell infiltration in the rabbit soleus (SOL) and gastrocnemius (GM) muscles in the exercised and non-exercised sides after 1 w, 3 w and 6 w of EMS/E.

<p>The values are presented as mean ± SD. Significant differences (p<0.05) are marked; * to controls,</p>▪<p>3 w <i>v.s.</i> 1 w group,</p>▴<p>6 w <i>v.s.</i> 1 w group and,</p>△<p>6 w <i>v.s.</i> 3 w group.</p

Data on primary antibodies used for detection of different MyHC isoforms.

<p>Data on primary antibodies used for detection of different MyHC isoforms.</p

Histological changes in the gastrocnemius muscle after 1

<p> <b>w and 6 </b><b>w.</b> Muscle samples from the exercised (left column, A, C) and non-exercised contralateral (right column, B, D) sides of gastrocnemius muscles after 1 w (A, B) and 6 w (C, D) of unilateral EMS/E. The sections are stained with H&E. Note the presence of atypical muscle fibers indicating muscle fiber regeneration in the exercised muscle (A) and fibers with internal nuclei (arrow) in the non-exercised muscle (B) after 1 w of EMS/E. Note also the large variation in the fiber size (A–D), the presence of small rounded fibers (C, D) and the marked fibrosis and inflammatory cell infiltration with extended duration of EMS/E (asterisks) (A, C, D) in both the exercised and non-exercised sides. Figure B and D shows presence of internal nuclei (arrows). (Bar = 50 µm).</p

Bilateral changes in vascular supply in affected areas in the exercised and non-exercised gastrocnemius and soleus muscles.

<p>Serial cross-sections from a control (A, F), an exercised (B, G) and non-exercised (C, H) gastrocnemius (GA) muscle and an exercised (D, I) and non-exercised (D, H) soleus (SOL) muscle. The cross-sections A–E are stained for mAb M0823 (CD31) against endothelium in vessels and the merged sections E–J are stained for CD31 (yellow/green), pAb PC128 against laminin (red) and DAPI (stains nuclei blue). Sections A, F show a control with a normal pattern of capillaries around fibers whereas section B–E, F–J shows different patterns of changes in capillarization in areas with inflammation and fibrosis. The samples from the exercised and non-exercised GA muscles shows examples of low capillary density in an area with inflammation/fibrosis (B, G) and in an area with mainly fibrosis (C, H). The sample from the exercised soleus muscle (D, I) shows an example of increased number of vessels with a larger size than capillaries in an area with fibrosis, inflammation and degenerating/regenerating fibers. The sample from the non-exercised SOL (E, J) shows an area with severe inflammation and increased capillarization. The differences in vascularization patterns probably represent different stages in the degenerative/regenerative process in the muscles. Arrows point at immunoreactions for capillaries and arrowheads against larger vessels in each section. Scale bar 50 µm.</p

Blood vessel distribution in normal and pathological muscle areas.

<p>The figure shows an example of blood vessel distribution in areas with normal morphology and in areas with pathological changes in the exercised gastrocnemius muscle after 3w of E/EMS. Section A is stained with HTX-eosin and shows an area with normal morphology (1), an pathological area with fibrosis and fat infiltration (2) and an area with severe mysositis and muscle fiber alterations (3). Sections B1-B3 are immunolabeled for polyclonal Ab PC128 against laminin, a component of the basement membranes of muscle fibers and vessels, and section C1–C3 are immunolabeled for mAb M0823 (CD31) against endothelium in vessels. Sections D1–D3 are merged images of immunolabeling for CD31 and laminin. Note the normal pattern of capillaries around fibers in the unaffected area (frame 1), the low number of capillaries in the area with fibrosis (frame 2), and the high number of comparatively larger vessels (arrows) in the area marked with inflammation (frame 3). Indications M1, M2 and M3 represent a serially sectioned muscle fiber in each frame stained for laminin (B), CD31 (C) and both laminin and CD31 (D). Scale bar 100 µm.</p

Serial sections of nerve fascicles.

<p>Cross-sections of nerves fascicles from the non-exercised side of the soleus muscle after 6w of EMS/E. The sections are stained with H&E (A, C) and for β-Tubulin (mAb T8660) (B, D). Framed region in (B) is inserted in larger magnification (top right). Stars show the corresponding area in (A, B) and (C, D). Note the weak or non-existing β-Tubulin immunoreaction for some axons in (B) and (D) (asterisks, marked with arrow in framed region). In (C), ballooned foamy cell structures are marked (arrows). Note also the fibrotic appearance and the presence of a large number of cell nuclei in the nerve fascicle in (C), especially in the area marked with a triangle. (Original magnification x200).</p