Effects of minced muscle tissue implantation and subsequent laser therapy on regeneration of skeletal muscles in guinea pigs]

It is known that regenerative capacity of skeletal muscles in guinea pigs is less than in rats. The guinea pig muscle regenerates are characterized by smaller muscle fibers and greater amounts of interstitial connective tissue. The present experiments were designed to stimulate the regenerative capacity of muscle tissue in adult guinea pigs, using tissue and laser therapy. It was shown that the minced muscle tissue implantation to the site of injury in m. gastrocnemius increased the quantity of regenerating muscle tissue. When the same operation was combined with following laser therapy the increase of muscle tissue was observed as well but not by far. At the same time laser therapy promoted noticeable acceleration of fibrin resorption, wound healing and regenerative modifications in implanted muscle fragments. Thus, the present methods stimulated relatively law regenerative capacity of guinea pig muscle tissue and promoted more qualitative recovery of injured area.     

Regenerative capacity of transplanted cardiac muscle in the rat

The authors analyzed the regenerative ability of cardiac muscle in the rat. Normal cardiac muscle was minced into mm3 fragments and homotransplanted into a triceps surae complex cavity of a nonsibling rat. Subsequent histological examination of the cardiac regenerate revealed the presence of myofibers. These myofibers typically exhibited centrally located nuclei, a characteristic of normal cardiac muscle. However, the absence of intercalated discs and autonomous contractility prevented indentification of these fibers as cardiac.     

Effects of autotransplantation of minced muscle tissue and subsequent laser therapy on recovery of the muscle injured by irradiation

A comparative histological investigation of posttraumatic regeneration in irradiated with 30 or 40 Gy and cross-sectioned musculus gastrocnemius of rats after autotransplantation into muscle defect of non-irradiated minced muscle tissue and laser therapy of hind limb in post-operative period was conducted. The obtained results showed that in irradiated with 30 Gy sectioned muscle (control series) the inflammatory reaction, resorption of fibrin in the area of trauma were inhibited and proliferation of muscle tissue from proximal and distal stump was suppressed. The rough connective tissue scar was formed. In experimental series for stimulation of regeneration the method of autografting minced muscle tissue into the defect of irradiated (30 or 40 Gy) cross-sectioned muscle and combination of this method with helium-neon laser rays exposition was used. The more marked recovery was obtained in irradiated with 30 Gy operated muscle after a 10-day treatment of limb with laser rays.     

Cell accumulation in the junctional region of denervated muscle

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.     

Repair and reorganization of minced cardiac muscle in the adult newt (Notophthalmus viridescens)

Studies of the response of adult mammalian and amphibian ventricle to injury have indicated the formation of a connective tissue scar in the place of the wounded or amputated muscle. It has been demonstrated that amphibian myocytes adjacent to a wound surface, unlike mammalian myocytes, have a proliferative capacity. In the present study, a minced cardiac muscle graft was placed into the adult newt ventricle in order to increase the number of myocytes near a wound surface. With such an increased number of reactive myocytes, it was thought a new wall consisting primarily of muscle might be formed. One-sixteenth to one-eighth of the ventricular apex was removed, minced and returned to the amputation surface of the ventricle. General histological and autoradiographic studies were conducted on two sham-operated animals and on five experimental animals which were killed at 5, 10, 20, 30, 50 and 70 days after surgery. Major events of the repair and reorganization of minced cardiac muscle included blood clot formation followed by necrosis of the blood clot and much of the muscle graft. By ten days, an apparent coalescence of muscle fragments and continuity of ventricular and graft lumina were observed, although the graft area never formed an integrated unit with the wounded ventricular wall. The peak of mitotic activity (3.19%) and thymidine labeling (28.1%) of graft cells, including many cells which resembled cardiac myocytes, was observed at 20 days. At 30 days, the graft was observed as a continuous wall composed primarily of muscle fibers. Several 30-, 50- and 70-day grafts had rhythmic contractions. These results suggest that amphibian cardiac muscle has histogenetic and proliferative capacities not attributable to mammalian cardiac muscle.     

The microscopic morphology of orthotopic free muscle grafts in rabbits

The flexor digitorum superficialis muscle was free grafted (without neurovascular anastomoses) in 122 rabbit forelimbs. Histologic nd histochemical examinations through 6 months after grafting were performed. An early ischemic necrosis of the entire graft, except for a few percent of fibers at the very surface, was consistently seen. Subsequently, there occurred a regeneration of muscle with reconstitution of up to 100 percent of normal numbers of fibers. There was a wide variation in the numbers of fibers regenerated; however, the fiber-free areas were then being replaced by connective tissue. Muscle grafts in 1-month-old rabbits regenerated faster and yielded muscle with evidence of more extensive reinnervation and less connective tissue than 3-month-old animals. In the early postgraft period, minced grafts appeared to be as good as whole ones, but after 1 month, they developed far more connective tissue. Differentiation into fast-twitch and slow-twitch muscle fibers and into high- and low-oxidative fibers began at 2 to 3 weeks after grafting but was not extensive until 3 months. At 6 months, grafts showed areas of normal-appearing muscle interspersed with areas that lacked signs of reinnervation. The earliest sign of regeneration is the appearance of several very elongated nuclei encircling each previously anucleate necrotic muscle fiber. A small amount of basophilic cytoplasm then appears around each new nucleus. As blood vessels grow into the graft, a centripetal wave of phagocytosis is seen, taking 2 to 3 weeks and leaving a bed of immature muscle fibers. We believe this to be the first documentation of regeneration's commencing prior to and thus independently of phagocytosis.     

Isoenzyme studies of whole muscle grafts and movement of muscle precursor cells

Summary Isoenzymes of glucose-6-phosphate isomerase (GPI: E.C. 5.3.1.9) were used as markers to determine the origin of cells which give rise to new muscle formed in allografts of whole intact muscle. GPI isoenzymes were also employed to see whether host precursor cells, which have been shown to contribute to muscle formation in grafts of minced muscle, can be derived from muscle lying adjacent to grafts.Excellent muscle regeneration was found in allografts of extensor digitorum longus (EDL) muscle examined after 58 days: 12 of 16 grafts contained 80% or more new muscle. Isoenzyme analysis showed that most, and in 2 instances all, new muscle was derived from implanted donor cells; however, there was strong evidence that in 5 grafts some, or all, new muscle must have resulted from host cells moving into the graft. Although hybrid isoenzyme was not detected this was attributed to factors associated with host tolerance which appear to interfere with fusion between host and donor myoblasts.Isografts of minced muscle were placed next to whole EDL muscle allografts to see if cells from allografts moved into adjacent regenerating tissue. Unfortunately, muscle regeneration in minced isografts was poor; only 3 contained 50% or more new muscle and most contained large amounts of fibrous connective tissue. Only a single isoenzyme band was detected in 11 isografts, but in five instances, the presence of a second band showed that cells from EDL allografts were also present. As no hybrid isoenzyme was detected, it is not known whether these cells which had moved into the regenerating minced grafts were muscle precursors, fibroblasts or some other cell types.     

Regeneration of the x-ray irradiated gastrocnemius muscle in old rats after stimulation

In the present study the implantation of nonexposed muscle tissue to the site of injury in irradiated musculus gastrocnemius and following laser therapy were applied in order to stimulate this muscle's posttraumatic regeneration in old rats. It was shown that a far larger amount of functionally active muscle tissue was formed at the site of injury compared with rats received laser therapy alone or the ones which were only implanted nonexposed minced muscle tissue. The muscle tissue consisted of muscle fibers which originated from the grafted pieces of nonexposed skeletal muscle and the ones produced by myofibers of muscle stumps recovered after irradiation. The connective tissue developed more evenly. The formation of adipose tissue was not observed at the site of injury. Moreover, the skin wound healing and the hair growth were stimulated as well.     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Muscle regeneration and the state of the thymus in adult rats under laser irradiation and alloplasty of newborn gastrocnemius muscle and diaphragm

The regeneration of adult rat gastrocnemius muscles has been studied under implantation of new-born rat muscles into area of muscle trauma. Alloplasty was performed using minced gastrocnemius and diaphragm muscles, which differs at birth in animals by degree of differentiation. The rat-recipient area of alloplasty was subjected to He-Ne laser radiation before operation, with the aim of reducing the immune response to allogenic muscle tissue. It has been shown that the number of regenerating myofibers produced from implanted gastrocnemius muscles is more than in alloplants from diaphragms. However, the formation of cartilage, bone, and adipose tissue foci was observed in the alloplastic region throughout the whole regeneration period. After implantation of minced diaphragm muscles, cartilage nodes were observed only in 7-day regenerates. At the end of observation, in the first instance, the area of muscle trauma in adult rat muscles was replaced by adipose tissue, even in the case of initial laser irradiation. After the implantation of diaphragm muscles, the area of trauma was filled with regenerating muscle tissue.     

The extracellular space of voluntary muscle tissues

The volume occupied by the extracellular space has been investigated in six types of voluntary muscles: sartorius (frog), semitendinosus (frog), tibialis anticus longus (frog), iliofibularis (frog), rectus abdominis (frog), and diaphragm (rat). With the aid of four types of probe material, three of which are conventionally employed (inulin, sorbitol, sucrose) and one of which is newly introduced (poly-L-glutamate), and a different experimental method, we have demonstrated that the "true" extracellular space of frog sartorius, semitendinosus, tibialis anticus longus, and iliofibularis muscle and of rat diaphragm muscle is equal to, or probably less than, 8-9% (v/w) of the tissue. The frog rectus muscle shows a somewhat higher ceiling value of 14%.     

Calcium equilibrium in muscle

1. A study of the calcium equilibrium in isolated frog muscle has been attempted. 2. When sartorius muscles were immersed in Ca(45) Ringer's solution, the surface phase took up the Ca(45) in about 1 minute; the extracellular water space and connective tissue in about 30 minutes; and the intracellular space in about 300 minutes. 3. The percentages of total calcium in the whole muscle immersed in Ringer's solution was as follows: 10 per cent in the surface phase; 12 per cent in the extracellular water space; 17 per cent in the dry connective tissue; 24 per cent in the intracellular space; and 37 per cent as non-exchangeable calcium. 4. The exchange constants of isolated frog sartorius muscle to calcium has been determined. The flux of intracellular calcium in the steady state was approximately 0.8 mM/(liter hr). 5. It appears that there is a calcium pump pushing calcium out of the cell against an electrochemical gradient of about 4 cal./mM of calcium. However, since the flux is low, the maximum energy required per hour to pump calcium out of the cell against this high gradient is only about 2 cal./kg. muscle or about 1 per cent of the resting energy.     

Synaptic activity and connective tissue remodeling in denervated frog muscle

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.     

Fenestrated blood capillaries and lymphatic capillaries in rat skeletal muscle

Capillary fenestrae occur in one of about 60 cross-sectioned blood capillaries in normal adult rat skeletal muscles. The fenestrae occur singly or in groups. Fenestrated capillaries are found close to muscle fibers as well as in the perimysial and perineurial connective tissue. Small numbers of lymphatic capillaries are also present, mostly in the perimysial connective tissue.     

Variability in the shapes of satellite cells in normal and injured frog sartorius muscle

Surface topography of satellite cells is examined in the scanning electron microscope after application of a new method to eliminate connective tissue components. We describe the overall shape of satellite cells in normal frog muscle and show the range of variation in normal structure. Small increases in the width of tails and changes in the cell outline occur after muscle injury in vivo and after incubation in vitro. These changes may be signs of early mobilization of satellite cells related to their eventual role in muscle regeneration.     

The suspensory muscle of the duodenum

The suspensory muscle of the duodenum continues the muscular layers of the duodenum, which is consequently surrounded on its mesenteric border only by the muscularis mucosae. Between the muscle bundles of the suspensor there is much loose connective and adipose tissue. Microganglions of no more than 90 cells may be found between the muscle bundles of the suspensor. In their superior part, the muscle bundles have connective sheaths, which are continuous with the adventitia of the aorta and celiac trunk. It is most probable that the suspensor acts like a sphincter and is innervated contrarily to the duodenum.     

Modeling of steady-state and relaxation elastic properties of the papillary muscle at rest

The biomechanical modeling of a papillary muscle preparation as an adequate object for studying the properties of the myocardial tissue under uniaxial stretching has been performed. The steady-state and relaxation tests of the papillary muscle of laboratory animals (rabbit and rat) have been conducted in normal conditions and after the maceration of intracellular structures with high ionic strength solution. It has been shown that the main contribution to the viscoelastic properties in the initial range of physiological deformations is made by the connective tissue skeleton, whereas under large physiological deformations, by intracellular structures.     

Fucose expression in skeletal muscle: a lectin histochemical study

Summary Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA. It is suggested that both the sarcoplasm and sarcolemma of diseased muscle fibres show altered fucose expression.     

The fine structure of smooth muscle cells and their relationship to connective tissue in the rabbit portal vein

Summary The smooth muscle of rabbit portal vein was studied by electron microscopy with particular emphasis on the mechanical linkage between the muscle cells and on the distribution of connective tissue.The media of this vein is composed of inner circular and outer longitudinal muscle layers which are orientated almost perpendicularly to each other. The muscle of the inner circular layer shows very irregular contours with much branching and anastomosing of the cytoplasmic processes, which often make membrane contacts with neighbouring cells to form an extensive network of cytoplasmic processes. The muscle cells of the outer longitudinal layer are arranged in densely packed bundles and are spindle-shaped, with no branching processes. Opposing dense areas from neighbouring cells, with variable gap distances (30–100 nm) and close membrane contacts (intermediate junctions) with a gap of 11 nm were observed in both circular and longitudinal muscle layers.In the terminal regions of muscle cells in both circular and longitudinal layers a specialized anchoring structure was present which was closely related to extracellular elastic tissue. Muscle cells in the longitudinal layer showed the most elaborate structure, the tapering end of the muscle cell showing a honeycomb-like structure penetrated by columns of connective tissue compounds. The functional implications of these structures are discussed.