RESUMEN
The proteolytic enzyme, chymase, was used to identify mast cells in rat gastrocnemius muscles which were crush-injured or incised in order to determine if mast cells exhibited proliferation and degranulation. Some of the crush-injured rats were subjected to 0 g for 14 days after injury on the Cosmos 2044 satellite to study the effects of weightlessness on the mast cell response. A variety of ground-based injured models were used, including a group of hindlimb-unloaded animals acting as controls and testing the suitability of the hindlimb-unloaded animal as a model for muscle healing during weightlessness. In most cases, the numbers of mast cells and their apparent size increased after injury. When mast cell degranulation was evident, the granules containing chymase often were free in the loose connective tissue and along the edge of myofibers. The mast cell response was most exaggerated in animals subjected to 0 g and least visible in the hindlimb-unloaded ones. Thus, gravitational stress may influence mast cell physiology and the hindlimb-unloaded animal may not be a good model for investigating muscle healing.
Asunto(s)
Gravitación , Mastocitos/patología , Músculos/patología , Animales , División Celular , Quimasas , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/ultraestructura , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Mastocitos/enzimología , Mastocitos/fisiología , Modelos Biológicos , Músculos/enzimología , Músculos/fisiología , Ratas , Ratas Sprague-Dawley , Federación de Rusia , Serina Endopeptidasas/análisis , Vuelo Espacial , IngravidezRESUMEN
The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on COSMOS 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various Earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with no enlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.
Asunto(s)
Matriz Extracelular/fisiología , Músculos/patología , Vuelo Espacial , Animales , Anticuerpos Monoclonales/inmunología , Capilares/ultraestructura , Permeabilidad Capilar , Concanavalina A/metabolismo , Endopeptidasas/metabolismo , Inmunohistoquímica , Masculino , Mastocitos/ultraestructura , Músculos/lesiones , Proteoglicanos/fisiología , Ratas , Ratas EndogámicasRESUMEN
The pathogenesis of dystrophin deficient myopathies remains unknown. Rat and human muscles subjected to severe injury following repeated eccentric muscle actions demonstrate histopathological alterations which mimic a dystrophic process. Immunofluorescent histochemical examination of these injured muscles demonstrates a separation of proteoglycans of the basal lamina from the muscle plasma membrane, the identical histopathological alteration observed in Duchenne muscular dystrophy. These findings are consistent with the hypothesis that dystrophin is essential for maintenance of the structural integrity of the sarcolemma.
Asunto(s)
Distrofina/fisiología , Músculos/lesiones , Distrofias Musculares/etiología , Distrofia Muscular Animal/fisiopatología , Adulto , Animales , Modelos Animales de Enfermedad , Estimulación Eléctrica , Humanos , Contracción Muscular , Músculos/patología , Músculos/fisiopatología , Distrofia Muscular Animal/etiología , Ratas , Ratas EndogámicasRESUMEN
Pain, stiffness, and indicators of muscle damage occur at different times after eccentric muscle action. After a single bout of maximal resisted lengthening of the elbow flexors, elbow position, pain perception, and indicators of cellular damage were measured. Immediately postexercise, a significant decrease in resting muscle length was observed that continued to 48 h. At this time, an increase in perceived muscle soreness was noted (P less than 0.05), and a biopsy of the biceps brachii revealed mast cell degranulation, separations of the extracellular matrix from myofibers, and increased plasma constituents in the extracellular space. It is proposed that myofiber disruption allows intracellular proteins to escape and extracellular proteins and ions to enter, causing swelling, whereas the disrupted extracellular matrix initiates the inflammatory response, which includes the release of mast cell granules seen at 48 h postexercise. Thus the delayed sensation of pain (soreness) after repeated eccentric muscle actions probably results from inflammation in response to extracellular matrix disruption.
Asunto(s)
Matriz Extracelular/patología , Músculos/fisiopatología , Dolor/fisiopatología , Adulto , Anciano , Anticuerpos Monoclonales , Biopsia , Sulfatos de Condroitina/metabolismo , Ejercicio Físico , Femenino , Humanos , Inmunohistoquímica , Masculino , Músculos/lesiones , Músculos/patología , Miositis/patología , Miositis/fisiopatología , Dolor/patología , Proteoglicanos/metabolismoRESUMEN
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.
Asunto(s)
Matriz Extracelular/patología , Músculos/lesiones , Heridas no Penetrantes/fisiopatología , Albúminas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Complemento C3/metabolismo , Tejido Conectivo/metabolismo , Tejido Conectivo/ultraestructura , Perros , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Fibrinógeno/metabolismo , Inmunohistoquímica , Inflamación , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Músculos/metabolismo , Músculos/fisiología , Proteoglicanos/inmunología , Proteoglicanos/metabolismo , Ratas , Ratas Endogámicas , Regeneración , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Músculos/lesiones , Animales , Técnica del Anticuerpo Fluorescente , Inflamación/metabolismo , Inflamación/patología , Linfocitos/patología , Lisosomas/enzimología , Macrófagos/patología , Masculino , Mastocitos/patología , Músculos/metabolismo , Músculos/patología , Péptido Hidrolasas/metabolismo , Ratas , Ratas Endogámicas , Regeneración , Estrés MecánicoRESUMEN
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.
Asunto(s)
Matriz Extracelular/metabolismo , Músculos/lesiones , Proteoglicanos/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Masculino , Músculos/metabolismo , Músculos/patología , Ratas , Ratas Endogámicas , Regeneración , Estrés Mecánico , Cicatrización de HeridasRESUMEN
A multi-catalytic protease in muscle cells was uniquely localized to the nucleus of muscle cells, both in cell culture and in sections of muscle tissue. Although no specific substructure of the nucleus could be identified as the site of the enzyme by immunocytochemical techniques, the enzyme was nevertheless present in all muscle cell nuclei. It appears that MCP is a useful marker for nuclei found specifically in muscle tissue.
Asunto(s)
Endopeptidasas/análisis , Músculos/citología , Animales , Núcleo Celular/enzimología , Núcleo Celular/ultraestructura , Inmunohistoquímica , Peso Molecular , Músculos/enzimología , Ratas , Ratas EndogámicasRESUMEN
Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.
Asunto(s)
Endopeptidasas/análisis , Músculos/enzimología , Animales , Células Cultivadas , Cisteína Endopeptidasas , Histocitoquímica , Inmunoquímica , Peso Molecular , Ratas , Ratas Endogámicas , Serina EndopeptidasasRESUMEN
Alkaline proteinase (chymase) was localized in skeletal muscle tissues from seven day streptozotocin-diabetic rats. Extruded mast cell granules containing proteinase were visible in the extracellular space and inside certain myofibers from both extensor digitorum longus (EDL) and soleus muscles. Additional diffuse staining was present in the cytoplasm of many EDL fibers. This evidence provides support for a possible role of muscle cells in the endocytosis of mast cell granules.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Endopeptidasas/análisis , Músculos/enzimología , Serina Endopeptidasas , Animales , Quimasas , Gránulos Citoplasmáticos/enzimología , Diabetes Mellitus Experimental/patología , Técnica del Anticuerpo Fluorescente , Mastocitos/enzimología , Ratas , Ratas Endogámicas , Coloración y EtiquetadoRESUMEN
The activity of four lysosomal proteases in soleus and extensor digitorum longus muscles was studied in streptozotocin-induced diabetic rats using newly developed fluorescence histochemical and biochemical techniques. The results indicate that the content of lysosomal protease in skeletal muscle cells was decreased three weeks after the induction of diabetes. The reduction was most pronounced in the extensor digitorum longus for all the proteases tested, but in the soleus only cathepsin B and dipeptidyl peptidase II showed a decrease. Biochemical assays on total muscle homogenates and muscle extracts confirmed the histochemical observations that protease activity was significantly lower in diabetic muscles. This decrease in activity varied with the duration of diabetes beginning as early as 48 h for the soleus. In conclusion, myofibre-specific decreases in lysosomal proteases occur following diabetes.
Asunto(s)
Catepsinas/metabolismo , Diabetes Mellitus Experimental/enzimología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Endopeptidasas/metabolismo , Lisosomas/enzimología , Músculos/enzimología , Animales , Diabetes Mellitus Experimental/patología , Cinética , Lisosomas/ultraestructura , Masculino , Microscopía Fluorescente/métodos , Músculos/patología , Ratas , Ratas EndogámicasRESUMEN
alpha 1-Antitrypsin and alpha 1-inhibitor-3 were localized for the first time inside skeletal muscle cells. Their content, especially that of alpha 1-inhibitor-3, was greatly reduced following streptozotocin-induced diabetes. alpha 1-Antitrypsin and alpha 1-inhibitor-3 were also observed in the vascular components and interstitial space surrounding both control and diabetic soleus muscles as revealed by immunofluorescence. In diabetic muscles, the non-myofibre locale of alpha 1-inhibitor-3 was reduced, and to a lesser extent, alpha 1-antitrypsin. Both myofibre and extracellular patterns were reversed to control levels by insulin replacement.