Survival of satellite cells in whole muscle transplants

The ability of satellite cells to survive the ischemic conditions at the core of orthotopically free grafted rat extensor digitorum longus muscles was examined. Cell cultures of isolated core and peripheral regions of whole muscle grafts maintained in vivo for more than 24 hours indicated that no viable cells were present in the core, whereas the number of cells from the peripheral region was greatly increased. Muscles were examined with the electron microscope to determine the fate of satellite cells of the core at various times after transplantation. The population of satellite cells in the core was reduced beginning at 18 hours and had virtually disappeared by 24-28 hours. This reduction did not appear to be the result of satellite cell death. Although there was abundant morphological evidence that myonuclei as well as myofiber cytoplasmic organelles were degenerating, there was little indication of satellite cell death in situ any time period studied. These studies suggest that satellite cells cannot survive, but migrate from the ischemic core to more peripheral regions of whole muscle transplants. In addition, they suggest that migration is an important aspect of the regeneration response in the free graft system and permits the myogenic population to contribute en masse to the centripetal wave of regeneration from the time it is initiated at the muscle periphery.     

Pathologic findings in long-term cardiac transplants

Since the introduction of cardiac transplantation at our institution 15 years ago, major advances have occurred in the monitoring and treatment of these patients, resulting in many long-term survivors. We defined the pathologic features in 14 cardiac transplants with survival times longer than one year. Only one heart showed no evidence of rejection, while the remaining 13 hearts showed advanced chronic rejection, which was the main cause of death or of graft failure in 11 patients. One patient died of gastric carcinoma, one of Kaposi's sarcoma, and one of cerebral embolus. The most obtrusive change in the donor hearts was an obliterative arteritis, which in the epicardial coronary arteries mimicked atherosclerosis. Superadded thrombosis often resulted in myocardial infarction. These severe vascular lesions bore no constant relationship to survival time and took from 1.1 to 12.5 years to evolve.     

Perisynaptic satellite cells in human external intercostal muscle: a quantitative and qualitative study

It is not known whether or not satellite cell nuclei are more common in the vicinity of motor endplates than in extrasynaptic regions of human muscle, as in animals. If so, perisynaptic satellite cells may have a role in preserving neuromuscular function. We compared the frequencies of satellite cell nuclei and of myonuclei in perisynaptic and extrasynaptic regions of human external intercostal muscle, and found an absolute as well as a relative increase of perisynaptic satellite cells. The mean frequency of satellite cell nuclei per sarcomere was 0.016 in perisynaptic and 0.00003 in extrasynaptic regions. The mean frequency of myonuclei per sarcomere was 0.098 in perisynaptic and 0.014 in extrasynaptic regions. We could not demonstrate any influence of aging on satellite cell distribution. Perisynaptic satellite cells had many processes, and some features suggested a more active state. These cells might add to the pool of junctional myonuclei for synthesis of acetylcholine-receptor molecules or help in the repair of the postsynaptic membrane. Alternatively, they may synthesize basal lamina substances that are specific for the endplate.     

The proximal attachments of the popliteus muscle: a quantitative study and clinical significance

Controversies about the existence of accessory proximal popliteus muscle attachments can be found in the literature. The aim of this study was to verify the occurrence and width of popliteus attachments on the articular and periarticular structures of the knee joint. The relation of these attachments to tibiofemoral cartilage and meniscus degeneration was also investigated. Forty-two anatomical specimens were dissected. The incidence of accessory proximal attachments was determined and their width measured using a caliper. The fibular attachment of the popliteus was observed in 98% of cases; its mean width was 11 (SD 3) mm. At least one attachment on the lateral meniscus was found in 95% of the specimens, with a mean width of 6 (SD 2) mm. Three types of meniscal attachments of the popliteus could be identified. The severity of meniscus and tibiofemoral cartilage alterations was significantly related to the number of meniscopopliteal fascicles: more severe alterations were seen in knees with fewer meniscopopliteal fascicles. Popliteus attachments on the posterior knee joint capsule (57%), arcuate (90%) and oblique popliteal (79%) ligaments were also observed in most specimens. Popliteus muscle relationships with the posterior cruciate (5%) and meniscofemoral (33%) ligament were less common. In conclusion, accessory popliteus attachments on the fibula, lateral meniscus and arcuate popliteal ligament can be considered constant characteristics. The results of this study suggest a role of the popliteus in the protection of knee menisci and tibiofemoral cartilage.     

Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The ultrastructure of myofibers in a reptilian heart: The boa constrictor

Electron microscopic observation of cardiac myofibers from the boa constrictor depict several features that differ from other vertebrate cardiac and striated muscle cells. The indentations in the sarcolemma do not penetrate into the deep sarcoplasmic matrix to form a transverse tubular system, instead the regular indentations extend the circumference of the myofiber at the regions of the Z line and are continuous with the latter via a dense homogeneous material that occurs at regular intervals along the subsarcolemmal surface. This dense material provides a direct contact between the sarcolemma and the contractile system. A complex sarcoplasmic reticulum was not observed around the myofibrils or in the sarcoplasmic matrix, instead there were small vesicles dispersed about the sarcoplasm. These myofibers are compared with the slow or tonic muscle fiber (Felderstruktur) which also lacks a transverse tubular system and contains an irregular sarcoplasmic reticulum.     

Myosatellite cells,growth, and regeneration in murine dystrophic muscle: A quantitative study

Patterns of growth and regeneration in 2-, 4-, 8-, and 17-week-old murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have been determined. Necrosis and myofiber loss, hypertrophy, and regeneration result in a reduced population of myofibers whose diameter distribution is more extensive than that found in the extensor digitorum longus muscles of age-matched normal mice. At the onset of dystrophic symptoms (2 weeks postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear population (myonuclei + myosatellite cells) is similar to that found in 2-week-old control muscles. The frequency of finding myosatellite cells decreases with age in both control and dystrophic muscles. Myosatellite cells account for 11%, 6%, 5%, and 3% of the total sublaminal nuclear population in control muscle and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in dystrophic muscle at 2, 4, 8, and 17 weeks, respectively. No preferential association of myosatellite cells with myofibers of a particular diameter is found in control muscle or in the two youngest dystrophic groups. At 8 and 17 weeks, myosatellite cells are less frequently encountered on small-diameter, regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of myosatellite cells decreases with age in both normal and dystrophic muscle. At all ages the myosatellite cell labeling index is higher in dystrophic muscle (23%, 7%, 5%, and 2% at 2, 4, 8, and 17 weeks, respectively) than in normal muscle (5%, < 1% at 2 and 4 weeks, respectively), with no labeled myosatellite cells being found in 8- and 17-week-old normal muscles. It is suggested that the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the regenerative response of dystrophic muscle to keep pace with the rapid muscle deterioration.     

Using a battery of tests to predict suicide in a long-term hospital: A quantitative analysis

Examined the Wechsler-Bellevue, Rorschach, TAT and Word Association Tests of 40 Patients for quantitative indications of suicide potential. Ten of these patients had completed suicide, 10 “serious” attempts, 10 “mild” attempts and 10 had never made a suicide attempt. In addition to a study of the quantification of major aspects of the tests, various published hypotheses also were examined. No quantitative aspect of the data differentiated all four groups. When the “completed” and “serious” attempters were compared to the “mild” and “no” attempters, the more lethal patients were found to give more movement responses and more total responses. Those who completed suicide also had a larger difference than others between their Verbal and Performance IQs. Of the published hypotheses, only the Roth & Blatt and Sapolsky hypotheses received significant support.     

Differences in fiber number and fiber type proportion within fascicles. A quantitative morphological study of whole vastus lateralis muscle from childhood to old age

Data are presented on the number of fibers and the proportion of different fiber types within fascicles of whole vastus lateralis muscles from 5 male children, 5 to 15 years of age, and compared with results from 25 male adults, middle aged and old individuals. The results verify a difference in the proportion of fibers with different properties within a fascicle. The proportion of type 2 (fast twitch) fibers on the border of fascicles is larger than the proportion internally: the children have (P<0.01) greater difference than the adults and the middle aged, whereas the old have (P<0.001) less difference than the other age groups. The mean number of fibers per fascicle increases (P<0.05) from childhood to adult age, and thereafter reduces (P<0.01). The results imply that fascicles continuously rearrange during the normal growth and development and the normal aging process. It is argued that the causes of the difference in fiber type proportions within fascicles are local factors in the muscle, secondary to the overall age related functional demands put on the fiber population.© Willey-Liss, Inc.     

Regional changes in the metabolite profile after long-term hypoxia-ischemia in brains of young and aged rats: a quantitative proton MRS study

Quantitative proton magnetic resonance spectroscopy (MRS) was used to determine region-specific metabolic changes in young and aged animals subjected to a long-term hypoxic-ischemic injury. Focal ischemia, which was studied as an experimental stroke model, was induced in 3- and 24-month-old rats by unilateral common carotid artery occlusion associated with 24 h of hypoxia. Eight metabolites were quantified from extracts in three different brain regions (hippocampus, frontoparietal and occipital cortices) from both the ipsilateral and contralateral sides. Our findings showed significant differences in lactate and myo-inositol concentration values in the hippocampus of the aged rats as compared to the same area of the young adult group under normoxic conditions. After hypoxia-ischemia (HI), the most relevant changes in metabolite concentrations were found in the hippocampal region of both young and aged groups as compared to their age-matched controls. Of the three brain areas under investigation, the hippocampus proved to be particularly susceptible to the prolonged hypoxia-ischemia perturbation. The effects were more evident in the aged animals.     

REM sleep and muscle atonia in brainstem stroke: A quantitative polysomnographic and lesion analysis study

Important brainstem regions are involved in the regulation of rapid eye movement sleep. We hypothesized that brainstem stroke is associated with dysregulated rapid eye movement sleep and related muscle activity. We compared quantitative/qualitative polysomnography features of rapid eye movement sleep and muscle activity (any, phasic, tonic) between 15 patients with brainstem stroke (N = 46 rapid eye movement periods), 16 patients with lacunar/non-brainstem stroke (N = 40 rapid eye movement periods), 15 healthy controls (N = 62 rapid eye movement periods), and patients with Parkinson's disease and polysomnography-confirmed rapid eye movement sleep behaviour disorder. Further, in the brainstem group, we performed a magnetic resonance imaging-based lesion overlap analysis. The mean ratio of muscle activity to rapid eye movement sleep epoch in the brainstem group (“any” muscle activity 0.09 ± 0.15; phasic muscle activity 0.08 ± 0.14) was significantly lower than in the lacunar group (“any” muscle activity 0.17 ± 0.2, p < 0.05; phasic muscle activity 0.16 ± 0.19, p < 0.05), and also lower than in the control group (“any” muscle activity 0.15 ± 0.17, p < 0.05). Magnetic resonance imaging-based lesion analysis indicated an area of maximum overlap in the medioventral pontine region for patients with reduced phasic muscle activity index. For all groups, mean values of muscle activity were significantly lower than in the patients with Parkinson's disease and polysomnography-confirmed REM sleep behaviour disorder group (“any” activity 0.51 ± 0.26, p < 0.0001 for all groups; phasic muscle activity 0.42 ± 0.21, p < 0.0001 for all groups). For the tonic muscle activity in the mentalis muscle, no significant differences were found between the groups. In the brainstem group, contrary to the lacunar and the control groups, “any” muscle activity index during rapid eye movement sleep was significantly reduced after the third rapid eye movement sleep phase. This study reports on the impact of brainstem stroke on rapid eye movement atonia features in a human cohort. Our findings highlight the important role of the human brainstem, in particular the medioventral pontine regions, in the regulation of phasic muscle activity during rapid eye movement sleep and the ultradian distribution of rapid eye movement-related muscle activity.     

Embryonic cord transplants in peripheral nerve restore skeletal muscle function

The rapid atrophy of skeletal muscle after denervation severely compromises efforts to restore muscle function. We have transplanted embryonic day 14-15 (E14-E15) ventral spinal cord cells into adult Fischer rat tibial nerve stump to provide neurons for reinnervation. Our aim was to evaluate medial gastrocnemius reinnervation physiologically because this transplant strategy will only be effective if the reinnervated muscle contracts, generates sufficient force to induce joint movement, and is fatigue resistant enough to shorten repeatedly. Twelve weeks posttransplantation, brief duration electrical stimuli applied to the transplants induced medial gastrocnemius contractions that were strong enough to produce ankle movement in 4 of 12 rats (33%). The force of these four "low-threshold" reinnervated muscles and control muscles declined only gradually during five hours of intermittent, supramaximal stimulation and without depression of EMG potential area, which is strong evidence of functional neuromuscular junctions and fatigue resistant muscles. Sectioning of the medial gastrocnemius nerves confirmed that these contractions were innervation dependent. Weakness in low-threshold reinnervated muscles (8% control force) related to incomplete reinnervation, reductions in muscle fiber size, specific tension, and/or the presence of nonfunctional neuromuscular junctions. Muscle reinnervation achieved using this novel transplantation strategy may salvage completely denervated muscle and may provide the potential to evoke limb movement when injury or disease precludes or delays peripheral axon regeneration.     

Succinic dehydrogenase activity in human muscle mitochondria during aging: a quantitative cytochemical investigation

A quantitative cytochemical study has been carried out on succinic dehydrogenase (SDH) activity in biopsy samples of vastus lateralis (VL) and anterior tibialis (AT) muscles from healthy men undergoing orthopaedic surgery. According to their age, the patients were divided into: young (25.0+/-4.4 years), middle-aged (50.4+/-7.5 years) and old (75.5+/-3.9 years) groups. Bioptically excised samples were processed for copper ferrocyanide preferential SDH cytochemistry. By a computer-assisted image analyser, we calculated the ratio (R): overall area of the precipitates due to the enzyme activity/area of each mitochondrion. No significant difference was found among the three age groups, despite an 8% increase of R in the adult vs. the other groups. R values are related to mitochondrial morphofunctional features since they may be modulated by enzyme activity and the physico-chemical conditions of the organelle membranes. Thus, R quantitation enables to estimate the mitochondrial capacities for adenosinetriphosphate provision. In this context, our present findings confirm previous data reporting a substantial age-related stability of muscle mitochondrial enzyme levels. In aging, energy-deficient sarcomeres are supported to be negatively selected and eliminated, while the surviving ones appear to maintain an adequate SDH activity.     

Macrophage infiltration in non-Hodgkin's lymphomas: a quantitative in situ study

The frequency and distribution pattern of macrophages within 93 non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry using stereological methods. For the identification of macrophages (M phi), several antibodies (Mono 1, Mono 2, OKM 1) reactive with surface antigens on cells of the monocyte-macrophage series and cytochemical staining for acid phosphatase were applied. The average number of macrophages within lymph node tissue of NHL was 6,299 +/- 760 cells/microliter (similar to reactive lymphatic tissue: 6,559 +/- 1,027). The highest number of infiltrating macrophages was detected in immunoblastic NHL (17,306 +/- 2,773), differing significantly from other histological subtypes and reactive lymphatic tissue (p less than 0.005). The possible impact of tumor-infiltrating macrophages on lymphoma cell proliferation and differentiation is discussed.