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

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

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

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

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

Localized histological changes in the muscle.

<p>Muscle sample stained with H&E from the exercised gastrocnemius muscle after 1 w of EMS/E. The figure shows a typical pattern of morphological changes and inflammation in local areas of the muscle tissue (bottom part). (Bar = 50 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

AChE reactivity pattern.

<p>Serial sections from a control (A, B) and a non-exercised (C, D) and exercised (E, F) soleus muscle after 6w of EMS/E. The sections are stained with H&E (A, C, E) and for AChE (B, D, F). A typical staining pattern for AChE in motor-endplates is shown in the control muscle (B) (arrows at corresponding locations in A and B). Figures D and F show an atypical AChE staining pattern of muscle fibers. Note the high AChE activity in the regenerating fiber in figure (D) (asterisks) and the AChE reaction on the surface of a fiber with normal morphology (arrow) in (F). A necrotic fiber is marked with an arrowhead in figures (C, D). Star marks similar fiber in the cross-sections. (Bar = 25 µm).</p

Staining for white blood cells.

<p>Muscle cross-sections from the non-exercised soleus muscle after 6w of EMS/E (A, B, D–G) and from the exercised side of the gastrocnemius muscle (C). The sections A–C are stained for demonstration of neutrophils/T-lymphocytes (mAb MCA805G). The framed region in (A) is in higher magnification in (B) and the parallel section to the framed region stained for H&E is in the inset (B). The figures show a large number of immuno-reactive cells (arrows) in the connective tissue (A–C). Stars show corresponding muscle fiber in (A) and (B). Figure D show infiltration of inflammatory cells in a necrotic fiber stained with H&E and figure E show the corresponding necrotic fiber (asterix) stained with mAb M0814 against CD68 (macrophages). Figure F show eosinophils (mAb MAB1087) infiltrating a muscle fiber (arrow) In (G), eosinophils (arrows) are stained with H&E in the extracellular matrix of another region of the muscle sample. (Original magnification; A ×100, B–E ×200, F and G x315).</p