William T. Stauber

Characterization of muscles injured by forced lengthening. I. Cellular infiltrates

Myofiber injury-repair was studied in rat soleus muscles to elucidate the role of infiltrating cells in the injury-repair process. Muscle injury was induced by forced muscle lengthening with the contralateral muscle serving as a control. The muscles were removed for histologic, histochemical and immunohistochemical procedures at varying periods (12-120 h) post-injury. All injured muscles were severely damaged with many cells present in the interstitial spaces between myofibers. Normal appearing myofibers demonstrated elevated lysosomal proteolytic activity, but no evidence of increased activity, indicative of phagocytic cells, was found in or between damaged myofibers. The esterase stain for macrophages and immunohistochemical techniques for mast cells also provided no support for either cell type predominating in the damaged area, although mast cell degranulation could be observed in the pericapillary regions. In contrast, the use of a specific antisera for a multicatalytic protease uniquely defined most of these cells as myogenic in origin. They appeared to be most numerous between the torn ends of a myofiber. Surprisingly, the remainder of the cells appeared to be of lymphoid origin.

Inhibition of lysosomal function in red and white skeletal muscles by chloroquine

Abstract In chickens treated 7 days with chloroquine, morphological observations and chemical analyses were in agreement with our hypothesis that lysosomes degrade some fraction of skeletal muscle mitochondria, plasma membrane, and glycogen but apparently not normal myofibrillar proteins. After chloroquine inhibition of lysosomal digestion, autophagy was apparent in anterior (ALD) and posterior (PLD) latissimus dorsi muscles. Membrane-limited vacuoles (autophagic vacuoles) contained mitochondria, membranes, and glycogen. To help identify the vacuolar constituents, a variety of marker enzyme activities and chemical analyses were monitored for the ALD and PLD muscles. Parallel increases in 5′-nucleotidase and cytochrome oxidase specific activities correlated with the increase in observable inclusions. Autolytic rates were increased in acidic ( p H 4.0) but not in alkaline ( p H 8.5) or neutral ( p H 7.0) conditions when homogenates were prepared from muscles of chloroquine-treated chickens. The removal of chloroquine inhibition in vitro by the preparation of tissue homogenates resulted in increased proteolysis by lysosomal enzymes presumably due to an increase in available substrates derived from mitochondria and plasma membranes.

Selective Extravascular Escape of Albumin into the Cerebral Cortex of the Diabetic Rat

The extravasation of plasma proteins (albumin, IgG, and complement C3) into the cerebral cortex was studied in streptozotocin-induced diabetic rats using im-munohistochemical techniques. The results indicate that albumin, but not IgG or complement C3, selectively enters the cerebral cortex within 2 wk after induction of diabetes. It is suggested that albumin may be an oncotic substance that contributes to diabetic cerebral microangiopathy, astrocytic swelling, and the cerebral edema that occasionally is seen during fluid and insulin therapy of juvenile ketoacidotic diabetes.

Cellular responses in exertion-induced skeletal muscle injury

Muscle injury is a common result of muscle exertion caused by overload and over-activity. In this presentation, an attempt was made to discuss models of muscle injury which involve exertion but not excessive strain, although most functional activities of the extremities require some eccentric muscle actions. Muscle injury is characterized by cellular and extracellular matrix responses which appear to be common to all types of muscle trauma - even in the absence of bleeding. Using tenotomy and functional over-load of the rat hindlimb muscles as examples, illustrations of several of these responses are presented and discussed.

Characterization of muscles injured by forced lengthening. II. Proteoglycans.

After forced muscle lengthening of rat soleus muscle, alterations in muscle connective tissues were monitored by fluorescent immunohistochemical methods. Monoclonal antibodies directed against the polysaccharide attachment region of proteoglycans were used to observe changes in localization of 4-sulfated, 6-sulfated, or unsulfated chondroitin sulfate disaccharide units covalently bound to the proteoglycan protein core after injury. Additionally, fluorescein-labeled concanavalin A lectin and polyclonal antiserum to heparan sulfate proteoglycan were also localized in muscle sections during the regenerative process over 5 days after injury. Although proteoglycan localization was absent at or near the site of myofiber damage after injury, some distinct basal lamina remained as a matrix for regenerating myofibers. By the fifth day post-injury, the localization of these matrix components had returned to that seen in uninjured soleus muscles. The physiological significance of these extracellular matrix changes appeared to center on the repair of the torn myofiber and indicate an interdependence between myofibers and the extracellular matrix in this type of regeneration.

Fluorescence Demonstration of Cathepsin B Activity in Skeletal, Cardiac, and Vascular Smooth Muscle'

Histochemical demonstration of cathepsin B activity was performed for the soleus, extensor digitorum longus, cardiac and vascular smooth muscle tissues of the rat using CBZ-Arg-Arg-4-methoxy-beta-naphthylamide or CBZ-Ala-Arg-Arg-4-methoxy-beta-naphthylamide as the substrate. The enzyme varied in its apparent activity but was localized in discrete granules in all muscle types. Cathepsin B was most active in cardiac muscle and least active in extensor digitorum longus muscles in between these extremes similar to another lysosomal protease, dipeptidyl peptidase II. However, in both types of skeletal muscle, the granules were observed more frequently at the periphery of the muscle cell just beneath the sarcolemma. Since cathepsin B is found only in lysosomes, this subsarcolemmal predominence may indicate that only one population of lysosomes in muscle contains active cathepsin B. All cathepsin B activity was abolished in the presence of the protease inhibitor, leupeptin.

Isometric and Concentric Performance of Electrically Stimulated Ankle Plantar Flexor Muscles in Intact Rat

The relationship between muscle force and ankle position during isometric and pre‐loaded slow concentric contractions (angular velocity, 0.52 rad s−1; range of motion, 1.22 rad) and the recovery of isometric force following concentric contractions at different velocities were determined for electrically stimulated plantar flexor muscles in intact rats. Pre‐loaded refers to the isometric contraction which immediately precedes the concentric contraction. Ankle position was controlled by a dynamometer and force was recorded under the sole of the foot. The peak isometric force (19.2 N) was nearly constant at all ankle positions (range of motion, 1.57 rad). The muscle length and distal fibre length of gastrocnemius medialis at ankle positions between 0.79 rad and 2.01 rad were increased by 12.6% and 20.3%, respectively. During slow concentric contractions, the force progressively decreased (23.1 ± 2.1%); the force decreased by only 6.3 ± 0.9% during sustained isometric contractions of similar duration (3400 ms). The recovery of isometric force following concentric contractions with similar stimulation frequencies (80 Hz) was velocity dependent (i.e. more rapid at higher velocities). It is concluded that pre‐loaded slow concentric contractions of the plantar flexor muscles in intact rats do not follow the same relationship as that of isometric force and ankle position. Our results in intact rats show that the force output of electrically stimulated ankle plantar flexor muscles measured under the sole of the foot can be used to study the physiological properties of skeletal muscle working in situ.

Recovery from 6 weeks of repeated strain injury to rat soleus muscles.

Recovery from chronic strain injury (50 strains daily, five times weekly for 6 weeks to hyperactive soleus muscles) was followed for 3 months in female rats after cessation of chronic hyperactivity induced by pretreatment of the plantar flexor muscles with tetanus toxin. After 6 weeks of repeated strains, muscle mass decreased by 62%, myofiber areas were reduced by 87%, and noncontractile tissue expanded dramatically by 222%. Collagen content increased by almost ninefold (control 40 ± 3 μg/mg, chronic injury 392 ± 53 μg/mg), whereas the molar ratio of collagen (pyridinoline) crosslinks to collagen remained the same (control 0.20 ± 0.01, chronic injury 0.16 ± 0.01). After 3 months of ambulation, muscle mass returned to normal but myofiber areas remained smaller by 21%, noncontractile tissue was still markedly elevated by 18% with increased collagen content (107 ± 15 μg/mg), and the molar ratio of crosslinks to collagen increased by 75% during recovery. Thus, rat soleus muscles recovered very slowly and incompletely from chronic strain injuries that produced muscle fibrosis, highlighting the necessity of devising preventative strategies for repeated strain injuries.

Extracellular matrix changes following blunt trauma to rat skeletal muscles

Myofiber injury-repair was studied in the rat following blunt trauma to the lower leg in order to understand how the inflammatory and regenerative responses of muscles are altered when myofiber rupture is accompanied by bleeding and clotting reactions. A contusion injury to the muscles of the lower hindlimb of the rat was induced by applying an impact force of 4.7 N-m/cm2 to one leg. The gastrocnemius and soleus muscles were removed bilaterally and evaluated by histochemical and immunohistochemical techniques to document myofiber, vascular, and connective tissue alterations for several days following insult (6-120 hr). A significant increase in wet weight of the gastrocnemius muscle was noted 24 hr postinjury as fluid accumulation and bruising were evident in the muscles resulting from bleeding and inflammation. Vascular disruption was confirmed by the localization of some plasma constituents (fibrinogen, albumin, and complement C3) throughout the interstitial space and even inside some of the damaged myofibers. Inflammation was present and persisted for 5 days as evidenced by continued mast cell degranulation and increased vascular permeability. Using antibodies to identify specific proteoglycans which appear or disappear at various times during muscle regeneration, muscle repair could be followed. The repair process required approximately 10 days for restoration of morphologically intact myofibers. Thus, myofiber repair processes appear to be maintained even after disruption of the vascular system and ischemia following blunt trauma.

The effect of four jaw relations on electromyographic activity in human masticatory muscles

Significant differences were found in the electromyographic (EMG) activation between the masseter and temporalis muscles for the leaf gauge (LG), manually manipulated (CR) and neuromuscular (NM) bite positions during maximal static clench. The LG position consistently demonstrated the lowest EMG activity, while the NM position displayed the highest degree of muscle activity. Similarly, the ratio of the masseter/temporalis EMG activity during maximal clench was lower for the LG and CR positions and highest for the NM position. These data indicate that the NM position produced the greatest total muscle recruitment, with more masseter involvement during maximal clench, and enabled the subjects to generate greater clenching forces in the NM position as compared to the LG and CR positions.