Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization

Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination.     

Effect of ecdysterone and juvenoid on the developmental involution of flight muscles in Acheta domestica

Morphogenesis and degeneration of the flight muscles in Acheta domestica was studied. The dorso-longitudinal flight muscles (DLMs) degenerate during the fourth day after adult ecdysis and the dorso-ventral flight muscles (DVMs) on the fifteenth day. In the presence of an intact innervation the degeneration of the DLMs can be retarded for 2 days by the injection of ecdysterone into very young adults. This retardation may also result in hypertrophy of the muscle fibres. The injection of ecdysterone, even in high doses, did not affect the flight muscle remnants. No notable changes have been found in the degeneration of DLMs by ovarectomy. Thus, the degeneration of flight muscles and the development of ovaries appear to be independent processes.The DLMs are homogeneous in fibre pattern in respect to succinic dehydrogenase, an important oxidative enzyme, and to ATPase activity, but the muscle fibres do not show any phosphorylase activity.     

Effects of short length immobilization of medial gastrocnemius muscle of growing young adult rats.

Effects of four and six weeks of immobilization at short length of gastrocnemius muscle on its architecture at optimum muscle length and length-force characteristics were studied. In general, immobilization effects were similar after 4 and 6 weeks. Smaller physiological cross-sectional area and lower muscle force were found as a consequence of immobilization. Muscle and aponeurosis were shorter. This was shown to be quantitatively related to atrophy i.e. smaller fibre diameter. Despite this atrophy no effects of immobilization on fibre and aponeurosis angles could be shown. Adaptation of the number of sarcomeres in series was found exclusively in distal fibres after 4 weeks of immobilization. No significant effects were found for proximal fibres of muscles at this time nor for any fibres after 6 weeks of immobilization. The effects of immobilization on muscle architecture did not affect the length range of active force exertion. It is concluded that muscle length adaptation as a consequence of short length immobilization is not related to adaptation of number of sarcomeres in series but to the occurrence of atrophy. It is also concluded that atrophy of pennate muscles does not have to be accompanied by a lower fibre and aponeurosis angle. Comparison of immobilized and control group rats indicates that the effects of immobilization can be characterized as a combination of retarded development of several variables and the influence of atrophy and its consequences.     

Extraocular muscle degeneration and regeneration after exposure of rats to incandescent radiant energy.

Exposure of albino rats to incandescent radiant energy for a short period of time in an elevated environmental temperature (39 degrees C) causes degenerative changes in the extraocular muscles. The muscle fibres regenerate and the muscles reorganize if the animals are returned to room lighting and temperature. Extraocular muscles (EOMs) were damaged first near their insertion on the eyeball. All EOMs of both eyes were affected, but the degeneration did not extend the entire length of the muscle. The peripheral fibres of each muscle were damaged before the more central fibres. Mitochondria were swollen and often contained dense bodies. Numerous vesicular profiles, possibly from the sarcotubular system, were present. Myofibrils of the more severely damaged fibres lacked typical Z-disk structures, and I-bands had disappeared by 24 h after the exposure period, a degenerative pattern which seems to be unique for this method of EOM damage. EOM degeneration appeared to be dependent on the interaction between thermal and radiant energy on the orbital contents. However, EOMs were only rarely and very slightly affected when rats were exposed to elevated temperature in the absence of incandescent radiant energy. When an opaque, black, ocular occluder was placed over one eye and the contralateral eye was left unoccluded, EOMs and retinas of occluded eyes were undamaged, while those tissues were severely damaged in unoccluded eyes. Therefore, the most critical single variable in inducing EOM degeneration appears to be exposure to radiant energy.     

Glycogen-enzymes containing bodies in type 2 fibres of tenotomized muscles in the rat

Inclusion bodies containing glycogen-enzymes were found in 30 to 60% of type 2 fibres of tenotomized calf muscles (m. gastrocnemius, m. soleus, m. plantaris) in rats, using histochemical reactions. The bodies appeared within 1 week after the tenotomy and were localized both in the central and the subsarcolemmal regions and rarely extruded into the extracellular space. These aggregates are 3 to 15 microns in length and 2 to 11 microns in diameter. In addition to glycogen, these bodies also contained various enzymes of the glycogen metabolism such as phosphorylase, a branching enzyme, and glucose-6-phosphatase, but showed no NADH-reductase, lactate dehydrogenase, or myofibrillar ATP-ase activity. The results indicate that glycogen-enzymes containing bodies are a degenerative phenomenon, which occurs only in type 2 fibres of the tenotomized muscles.     

Acute Rhabdomyolysis (“Tying-Up”) in Standardbred Horses

LINDHOLM, A., H.-E. JOHANSSON & P. KJÆRSGAARD: Acute rhabdomyolysis (“tying-up”) in standardbred horses. A morphological and biochemical study. Acta vet. scand. 1974, 15, 325–339. — Morphological, biochemical and histochemical changes were studied in muscle needle biopsy specimens (gluteus medius) from 59 standardbred trotters with acute clinical symptoms of the “tying-up” disease. All horses had increased levels of serum enzymes SGOT and SCPK. The biopsy specimens were taken at various intervals after onset of clinical symptoms (1–4 hrs., 18–24 hrs. and 2–20 days). Ry light microscopy it was shown that the muscular alterations had a focal distribution and were of the hyalin degeneration type with insignificant inflammatory reaction and slight calcification. The ultrastructural changes apparently commenced with myofibrillar waving, mitochondrial and sarcotubular alterations and terminated with myofibrillar degeneration and necrosis with invasion of inflammatory cells. The inflammatory cells were ultrastructurally similar to monocytes and macrophages. The degenerative changes mainly comprised fast twitch (FT and FTH) fibres as histochemically evidenced by myofibrillar ATPase and alkaline phosphatase staining. Riopsies from diseased muscle 1–4 hrs. after the onset of “tyingup” contained a low muscle concentration of glycogen, ATP and CP and a high concentration of lactate and glucose. Hence it is suggested that the described muscular alterations may be caused by a deranged carbohydrate metabolism caused by a local hypoxia. It was found that the “tying-up” disease resembled idiopathic rhabdomyolysis in man and was thus designated “equine rhabdomyolysis”. histochemistry; horse; rhabdomyolysis; skeletal muscle; “tying-up”; ultrastructure.     

Short-term immobilization and recovery affect skeletal muscle but not collagen tissue turnover in humans

Not much is known about the effects of immobilization and subsequent recovery on tendon connective tissue. In the present study, healthy young men had their nondominant leg immobilized for a 2-wk period, followed by a recovery period of the same length. Immobilization resulted in a mean decrease of 6% (5,413 to 5,077 mm(2)) in cross-sectional area (CSA) of the triceps surae muscles and a mean decrease of 9% (261 to 238 N.m) in strength of the immobilized calf muscles. Two weeks of recovery resulted in a 6% increased in CSA (to 5,367 mm(2)), whereas strength remained suppressed (240 N.m). No difference in Achilles tendon CSA was detected between the two legs at any time point. Local tendon collagen synthesis, measured as the peritendinous concentrations of PINP (NH(2)-terminal propeptide of type I collagen; indirect marker for collagen synthesis), was unchanged after 2 wk of immobilization. However, peritendinous levels of PINP were significantly elevated in the immobilized leg (15 to 139 ng/ml) following 2 wk of remobilization compared with preimmobilization levels. In contradiction hereto, systemic concentrations of PINP remained unchanged throughout the study. Immobilization reduced muscle size and strength, while tendon size and collagen turnover were unchanged. While recovery resulted in an increase in muscle size, strength was unchanged. No significant difference in tendon size could be detected between the two legs after 2 wk of recovery, although collagen synthesis was increased in the previously immobilized leg. Thus 2 wk of immobilization are sufficient to induce significant changes in muscle tissue, whereas tendon tissue seems to be more resistant to short-term immobilization.     

Protein synthesis in adult skeletal muscle after tenotomy: responses to fasting and insulin infusion.

Muscle growth was established in specific muscles in the hindlimb of adult female rats by tenotomy of the gastrocnemius muscle. Seven days after surgery there was an increase in the wet weight of the soleus (Sol) and plantaris (P) muscles and a decrease in that of the gastrocnemius (G) muscle from the tenotomized limb compared with the respective control muscles from the contralateral limb from the same animal. In all three muscles there was a significant increase in the fractional rate of protein synthesis (ks) in the muscles from the tenotomized limb above the rate of the respective control muscles. In contrast, the extensor digitorum longus (EDL) muscle showed no change in wet weight or ks 7 days after tenotomy of G. Fasting for 12 or 36 h had no significant effect on ks in G, P, or Sol muscles from either the control or tenotomized limbs. In EDL from the control limb, both fasting periods resulted in a significant decrease in ks, although this effect was not seen in the EDL from the tenotomized limbs of the same animals. A subsequent 30-min insulin infusion was similarly ineffectual in G, P, and Sol, with its only effect evident in the EDL from the control limb, where it was sufficient to reverse the decreased ks resulting from the fasting, even though after 36 h fasting the reversal was only partial.     

Effects of dietary cobalt on testicular structure

Adult male rats were maintained on a diet containing 265 ppm cobalt for up to 98 days. Three rats were sacrificed weekly and assayed for testicular damage by light and electron microscopy. Testicular damage was first apparent after 70 days of treatment, followed by a progressive deterioration of cell architecture and decrease in testicular volume. The degenerative changes were of a very general nature; e.g., thickening of basal lamina and basement membranes, increased packing of red blood cells in veins and arteries, formation of "giant" cells, loss of sperm tail filaments, and degeneration of sperm mitochondria. No cobalt residues could be detected by energy dispersive x-ray microanalysis. These data indicate that testicular degeneration was not a primary response to cobalt and suggest that the testes become hypoxic due both to blockage of veins and arteries by red blood cells and to changes in permeability caused by thickening of basal lamina and basement membranes.     

小鼠骨胳肌在切腱、协同肌切腱和肉毒杆菌毒素中毒后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用生物化学方法测定离体小鼠比目鱼肌对 HRP 的胞纳作用。结果表明,在切断神经或切腱后引起萎缩的肌肉侧或在协同肌切腱后引起代偿性肥大的肌肉侧,与它们各自对照的正常肌肉侧相比,都可发生对 HRP 胞纳的明显增加。而在肉毒杆菌毒素中毒后引起萎缩的肌肉侧,和它正常的对照肌肉侧相比,却意外地不发生这种胞纳摄取的明显增加。本文的实验结果进一步证实。肌肉的胞纳增加并不一定导致肌纤维的变性和萎缩(例如代偿性肥大的肌肉);而肌纤维的变性和萎缩亦不一定需要肌肉的胞纳增加为前提(例如肉毒杆菌毒素中毒的肌肉)。本工作结果还提示:肌肉的胞纳增加有其神经原性和肌原性因素。本文还对肌肉胞纳增加的可能机制进行了讨论。     

饥饿和交配对小地老虎飞行肌发育的影响

小地老虎Agrotis ypsilon(Rottemburg)成虫飞行肌的发育常受一些因素影响而发生变化,为探讨饥饿和交配行为对飞行肌发育的影响,通过电子显微镜对雌虫飞行肌(背纵肌)的肌原纤维、线粒体结构进行观察,结果显示:4日龄饥饿雌虫,肌原纤维直径、肌节长度、肌原纤维体积均显著(P<0.05)小于取食的。7日龄饥饿雌虫肌原纤维直径、肌节长度、肌原纤维体积分数较4日龄的差异均不显著(P≥0.05),而7日龄饥饿的肌原纤维直径显著(P<0.05)大于7日龄取食的;羽化10 d后,饥饿雌虫肌节长度显著(P<0.05)大于取食雌虫的,而肌纤维体积分数和线粒体体积分数均却小于后者。7、10、13日龄交配雌虫肌原纤维横切直径分别显著(P<0.05)小于同日龄非交配的;7、10、13日龄交配雌虫肌原纤维体积分数显著(P<0.05)小于非交配的,线粒体体积分数虽然无差异(P≥0.05),但是交配雌虫的早在4日龄便已明显(P<0.05)减小。上述结果表明:正常取食的小地老虎飞行肌4日龄后会发生降解现象;饥饿抑制飞行肌前期发育和中期的降解,而促进成虫末期肌原纤维的分解;交配能促进飞行肌的降解。     

Degenerate Human Nucleus Pulposus Cells Promote Neurite Outgrowth in Neural Cells

Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.     

Tenotomy of the avian anterior latissimus dorsi muscle. II. Can regeneration from the stump occur in the pigeon?

Because the chick's anterior latissimus dorsi muscle (ALD) regenerates a fast-twitch muscular connection after tenotomy, the pigeon's ALD was tenotomized, either at the origin or through the muscle 0.5 cm from the origin, to determine whether this muscle behaves similarly to the chick muscle. These procedures were compared in pigeons operated upon at 7 weeks, versus 5 to 9 months of age. The pigeon's ALD did not regenerate a new connection, and other differences were observed between the pigeon and chick ALD. The pigeon ALD has only a single slow muscle-fiber type, has fewer fast fibers, and transforms to a fast-twitch muscle more readily than the chick ALD after tenotomy. The transformation of muscle fiber types occurred more readily in the older pigeons than those tenotomized at 7 weeks of age. Tenotomy induced morphological alterations of the muscle fiber structure in all of the pigeons, which is in contrast to the absence of changes in the tenotomized chick ALD. Therefore the pigeon and chick ALD respond completely differently to tenotomy.     

Evidence that myenteric neurons of the gastric corpus project to both the mucosa and the external muscle: myectomy operations on the canine stomach

Summary The distribution of nerve cell bodies and fibres in the canine stomach was investigated using antibodies to the general neuronal marker, neuron-specific enolase. Prominent ganglia containing many reactive nerve cells were found in the myenteric plexus of the gastric corpus and antrum. Nerve cells were absent from the submucosa of the corpus and were extremely rare in the antrum. Renoval of areas of longitudinal muscle and myenteric plexus from the corpus (myectomy), with 7 days allowed for axon degeneration, resulted in the loss of fibres reactive for galanin, gastrin-releasing peptide, substance P and vasoactive intestinal peptide from both the circular muscle and mucosa in the area covered by the lesion. Combined vagotomy and sympathetic denervation did not significantly affect these fibres, but did cause fibres reactive for calcitonin gene-related peptide to degenerate. It is concluded that the myenteric plexus of the gastric corpus, like the myenteric plexus of the small intestine and colon, is the source of nerve fibres innervating the circular muscle, but, in contrast to other regions of the gastrointestinal tract, myenteric ganglia, not submucous ganglia, are the major, or sole, source of the intrinsic innervation of the mucosa.     

Ultrastructural degenerative changes of age-dependent ECHO 9 virus-induced polymyositis in infant mice

Experimental inoculation of newborn NMRI mice with Echo virus, type 9, strain A. Barty *ECHO 9 virus AB) resulted in diffuse polymyositis 4 days later. The disease, after a delay of 1 day, increased in intensity and was accompanied by a rapid, progressive paresis leading to death 7-11 days following infection. As early as 24-48 h after inoculation, we detected initial ultrastructural degenerative changes, characterized by segmental dilation fo the sarcoplasmic reticulum. Disruption and breakage of myofibrils followed, and lead to the formation of increasing numbers of contraction bands. Ultimately, virus-mediated nuclear and other organellar injury resulted in muscle fiber necrosis. In addition, we observed some substructural cellular events related to virus propagation. Beginning on the 4th day after infection, pronounced proliferation of the sarcoplasmic-reticulum membranes became evident in perinuclear and subsarcolemmal areas in the mature muscle fibers, myoblasts, and post-fused myotubes of mice inoculated within 18 h after birth. These cytological alterations were not noted in the myoblasts and myotubes of NMRI mice injected with virus 2 or 4 days after birth.     

Autotransplantation of previously denervated muscles in the rabbit

Whole gastrocnemius muscles of rabbits, preliminarily denervated, were grafted. At the moment of grafting (60 days after the operation) the muscles were in the state of deep atrophy attended by distrophic changes.The autotransplantated muscles took at the site of grafting, their further reorganization provided progressive development of the muscle tissue within the transplant, its growth, and formation of definitive muscle fibers with nerve terminals. After a definite time some degenerative changes were observed in the transplant muscle tissue; as a result the muscle tissue was substituted by connective tissue. These data support the statement founded before on feasible free grafting of preliminary denervated whole muscles. However, deep denervation atrophy seems to influence the remote results of the transplantation.     

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle. A histochemical study.

Adult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, beta-glucuronidase, beta-N-acetylglucosaminidase, and beta-glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of beta-glucuronidase and beta-N-acetylglucosaminidase was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3--7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue. beta-Glycerophosphatase activity was not, by the method used, seen in histologically "healthy" or normal-looking fibres. In samples taken 2--5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which possess features typical of both lethal and sublethal acute cell injury.     

Effect of transection at different levels of hypothalamus on the hypothalamo-hypophysial system of the toad,Bufo bufo,with particular reference to the ultrastructure of the zona externa of the median eminence

Summary The ultrastructural changes taking place in the median eminence of Bufo bufo 3 hours to 4 months after transection of the brain at different levels, are described.5 types of neurons in the zona externa of the median eminence of normal toads are described. All types of neurons degenerate, and profound changes of the ultrastructure of the capillaries are observed after transection just behind, or immediately in front of the optic chiasma. A few neurons containing dense granules with a diameter of about 1,000–1,300 Å remain intact, however. The degeneration following denervation in front of the optic chiasma was considerably delayed compared to degeneration after denervation behind the optic chiasma.After transection more rostral to the optic chiasma, no significant degeneration of the median eminence was observed.The results are discussed with regard to degenerative dynamics and origin of the different nerve types. It is concluded, that all types of neurons terminating in the median eminence, originate at a level between the caudal and rostral parts of the preoptic nucleus, some fibres, however, containing dense, 1,000–1,300 Å granules, originate caudally to the optic chiasma, in the posterior hypothalamus.Part of this study was presented at the Vth International Symposium on Neurosecretion, Kiel, Germany, August 1969.     

Hindlimb immobilization applied to 21-day-old mdx mice prevents the occurrence of muscle degeneration.

Dystrophin-deficient skeletal muscles of mdx mice undergo their first rounds of degeneration-regeneration at the age of 14-28 days. This feature is thought to result from an increase in motor activity at weaning. In this study, we hypothesize that if the muscle is prevented from contracting, it will avoid the degenerative changes that normally occur. For this purpose, we developed a procedure of mechanical hindlimb immobilization in 3-wk-old mice to restrain soleus (Sol) and extensor digitorum longus (EDL) muscles in the stretched or shortened position. After a 14-day period of immobilization, the striking feature was the low percentage of regenerated (centronucleated) myofibers in Sol and EDL muscles, regardless of the length at which they were fixed, compared with those on the contralateral side (stretched Sol: 8.4 +/- 6.5 vs. 46.6 +/- 10.3%, P = 0.0008; shortened Sol: 1.2 +/- 1.6 vs. 50.4 +/- 16.4%, P = 0.0008; stretched EDL: 05 +/- 0.5 vs. 32.9 +/- 17.5%, P = 0. 002; shortened EDL: 3.3 +/- 3.1 vs. 34.7 +/- 11.1%, P = 0.002). Total numbers of myofibers did not change with immobilization. This study shows that limb immobilization prevents the occurrence of the first round of myofiber necrosis in mdx mice and suggests that muscle contractions play a role in the skeletal muscle degeneration of dystrophin-deficient mdx mouse muscles.