New tools for the investigation of muscle fiber-type spatial distributions across histological sections.

BACKGROUND: The functional and metabolic properties of skeletal muscles are partly a function of the spatial arrangement of fibers across the muscle belly. Many muscles feature a non-uniform spatial pattern of fiber types, and alterations to the arrangement can reflect age or disease and correlate with changes in muscle mass and strength. Despite the significance of this event, descriptions of spatial fiber-type distributions across a muscle section are mainly provided qualitatively, by eye. Whilst several quantitative methods have been proposed, difficulties in implementation have meant that robust statistical analysis of fiber type distributions has not yielded new insight into the biological processes that drive the age- or disease-related changes in fiber type distributions. METHODS: We review currently available approaches for analysis of data reporting fast/slow fiber type distributions on muscle sections before proposing a new method based on a generalized additive model. We compare current approaches with our new method by analysis of sections of three mouse soleus muscles that exhibit visibly different spatial fiber patterns, and we also apply our model to a dataset representing the fiber type proportions and distributions of the mouse tibialis anterior. RESULTS: We highlight how current methods can lead to differing interpretations when applied to the same dataset and demonstrate how our new method is the first to permit location-based estimation of fiber-type probabilities, in turn enabling useful graphical representation. CONCLUSIONS: We present an open-access online application that implements current methods as well as our new method and which aids the interpretation of a variety of statistical tools for the spatial analysis of muscle fiber distributions.

Use of a novel technique to assess impact of age-related denervation on mouse soleus muscle function.

Denervation contributes to loss of force-generating capacity in aged skeletal muscles, but problems with quantification of denervated fibers mean the precise impact of denervation on muscle function remains unclear. This study therefore looked to develop a reliable assay for identifying denervated muscle fibers, and used this to explore the impact of denervation on age-related force-generation in mouse skeletal muscle. Thirteen young (6-month-old) and 10 old (24-months-old) C57Bl/6 J female mice were utilized. Anaesthetized mice were infused with the fluorescent deoxyglucose analog 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) and the tibial nerve was repeatedly stimulated to label active skeletal muscle fibers by activity-dependent uptake of 2-NBDG. Data on muscle force generation were acquired as part of the stimulation routine. Labeled muscles were removed, snap frozen, sectioned, and slide mounted. Sections were imaged to show accumulation of 2-NBDG in activated fibers and lack of 2-NBDG accumulation in quiescent (denervated) fibers, then processed using immunohistochemistry to allow collection of data on fiber number and morphology. Soleus muscles from older mice had nine times as many denervated fibers as those from young mice (average n = 36 vs 4, old vs young). Older muscles developed significantly more passive force and less specific force, but denervation only partly accounted for age-related deficits in specific force. Further investigations are required to definitively identify contributors to the decrease in force generation that remain unaccounted for.

Age-related structural changes show that loss of fibers is not a significant contributor to muscle atrophy in old mice.

Age-related loss of skeletal muscle mass is widely considered a consequence of both fiber atrophy and fiber death. Evidence for fiber death derives largely from an age-related reduction in fiber numbers in muscle cross-sections, however it is unclear how age-related alterations in muscle morphology affect accuracy of such counts. To explore this we performed an examination of muscle and tendon length, muscle mass and girth, and pennation angle, in addition to histological section fiber counts of parallel-fibered (sternomastoid), fusiform (biceps brachii), and pennate (tibialis anterior, extensor digitorum longus, soleus) muscles from 31 mice aged 6-32 months. Age-related decline in mass and girth occurred in soleus (p = 0.026; p = 0.040), tibialis anterior (p = 0.004; p = 0.039), and extensor digitorum longus (p = 0.040; p = 0.022) muscles, for which location of maximal girth also changed. Tendon length and pennation angle remained consistent across the lifespan in all except soleus which showed elongation of both proximal and distal tendons coupled with alterations in pennation angle. Age-related decreases in fiber number were observed in transversely sectioned soleus and extensor digitorum longus muscles however when age-related changes in morphology were accounted for via oblique sectioning the age-related decrease in fiber number was eliminated. Findings show loss of fibers is not a significant contributor to age-related muscle wasting in mice, and that age-related changes in connective tissue selectively impact muscle structure. Fiber shortening is a likely contributor to loss of mass and change in function in muscles of old mice.

Increased nuclear permeability is a driver for age-related motoneuron loss.

Sarcopenia is the loss of skeletal muscle mass with age, the precise cause of which remains unclear. Several studies have shown that sarcopenia is at least partly driven by denervation which, in turn, is related to loss of motor nerve cells. Recent data suggests degradation of the nucleocytoplasmic barrier and nuclear envelope transport process are contributors to nerve loss in a number of neurodegenerative diseases. Having recently shown that important components of the nuclear barrier are lost with advancing age, we now ask whether these emergent defects accompany increased nuclear permeability, chromatin disorganization and lower motoneuron loss in normal ageing, and if so, whether exercise attenuates these changes. Immunohistochemistry was used on young adult, old and exercised mouse tissues to examine nucleocytoplasmic transport regulatory proteins and chromatin organization. We used a nuclear permeability assay to investigate the patency of the nuclear barrier on extracts of the spinal cord from each group. We found increased permeability in nuclei isolated from spinal cords of old animals that correlated with both mislocalization of essential nuclear transport proteins and chromatin disorganization, and also found that in each case, exercise attenuated the age-associated changes. Findings suggest that the loss of nuclear barrier integrity in combination with previously described defects in nucleocytoplasmic transport may drive increased nuclear permeability and contribute to age-related motoneuron death. These events may be significant indirect drivers of skeletal muscle loss.

Exercise attenuates age-associated changes in motoneuron number, nucleocytoplasmic transport proteins and neuromuscular health.

Life expectancy continues to extend, although frailty caused by loss of skeletal muscle mass continues unimpeded. Muscle atrophy caused by withdrawal of motor nerves is a feature of old age, as it is in amyotrophic lateral sclerosis (ALS) in which skeletal muscle denervation results from motoneuron death. In ALS, direct links have been established between motoneuron death and altered nucleocytoplasmic transport, so we ask whether similar defects accompany motoneuron death in normal ageing. We used immunohistochemistry on mouse tissues to explore potential links between neuromuscular junction (NMJ) degeneration, motoneuron death and nucleocytoplasmic transport regulatory proteins. Old age brought neuromuscular degeneration, motoneuron loss and reductions in immunodetectable levels of key nucleocytoplasmic transport proteins in lumbar motoneurons. We then asked whether exercise inhibited these changes and found that active elderly mice experienced less motoneuron death, improved neuromuscular junction morphology and retention of key nucleocytoplasmic transport proteins in lumbar motoneurons. Our results suggest that emergent defects in nucleocytoplasmic transport may contribute to motoneuron death and age-related loss of skeletal muscle mass, and that these defects may be reduced by exercise.

Age-related remodelling of the myotendinous junction in the mouse soleus muscle.

The age-related loss of muscle mass and function predominantly affect muscles of the lower limbs and have largely been associated with decline in muscle fibre size and number, although the exact mechanisms underlying these losses are poorly understood. In addition, consistent reports that the loss of muscle strength exceeds that which can be explained by declines in muscle mass has widened the search for causes of sarcopenia to include supporting tissues such as the extracellular matrix and tendons. Although the changes to both muscle and tendon with age are well characterised, little work has focused on the interface between these two tissues, the myotendinous junction (MTJ). Given the crucial role for this structure in force transfer between muscle and tendon, we asked whether the myotendinous junction underwent structural changes with age in lower limb muscle. We used whole muscle to assess gross muscle and tendon morphology, and immunohistochemistry to determine fibre and MTJ profile number in young (6 months), middle aged (18 months) and elderly (24 months) C57BL/6 female mice. MTJ length was quantified using serial cross sections of the soleus muscle. We found an apparent 3.5-fold increase in MTJ profiles per cross section with no increase in fibre number in old mice, and found this to be a result of a doubling in length of the MTJ region with age. This coincided with an increase in proximal tendon length (31%), as well as an increase in collagen deposition between 6 and 24-months of age consistent with an expansion of the fibre termination area. These findings uncover a previously undescribed effect of ageing on the MTJ and open up new lines of investigation into the role of this structure in the age-related loss of muscle function.

IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice.

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.

Elderly mouse skeletal muscle fibres have a diminished capacity to upregulate NCAM production in response to denervation.

Sarcopenia is a major contributor to the loss of independence and deteriorating quality of life in elderly individuals, it manifests as a decline in skeletal muscle mass and strength beyond the age of 65. Muscle fibre atrophy is a major contributor to sarcopenia and the most severely atrophic fibres are commonly found in elderly muscles to have permanently lost their motor nerve input. By contrast with elderly fibres, when fibres in young animals lose their motor input they normally mount a response to induce restoration of nerve contact, and this is mediated in part by upregulated expression of the nerve cell adhesion molecule (NCAM). Therefore, skeletal muscles appear to progressively lose their ability to become reinnervated, and here we have investigated whether this decline occurs via loss of the muscle's ability to upregulate NCAM in response to denervation. We performed partial denervation (by peripheral nerve crush) of the extensor digitorum longus muscle of the lower limb in both young and elderly mice. We used immunohistochemistry to compare relative NCAM levels at denervated and control innervated muscle fibres, focused on measurements at neuromuscular junctional, extra-junctional and cytoplasmic locations. Muscle fibres in young animals responded to denervation with significant (32.9%) increases in unpolysialylated NCAM at extra-junctional locations, but with no change in polysialylated NCAM. The same partial denervation protocol applied to elderly animals resulted in no significant change in either polysialylated or unpolysialylated NCAM at junctional, extra-junctional or cytoplasmic locations, therefore muscle fibres in young mice upregulated NCAM in response to denervation but fibres in elderly mice failed to do so. Elevation of NCAM levels is likely to be an important component of the muscle fibre's ability to attract or reattract a neural input, so we conclude that the presence of increasing numbers of long-term denervated fibres in elderly muscles is due, at least in part, to the fibre's declining ability to mount a normal response to loss of motor input.

Dying myofibers in elderly mouse skeletal muscles are characterized by the appearance of dystrophin-encircled vacuoles.

The age-related loss of skeletal muscle mass and strength (sarcopenia) is predominantly attributed to myofiber atrophy, however the role or existence of myofiber death is currently unclear. We recently discovered dysmorphic myofibers in normal elderly mice resembling those that characterize the Autophagic Vacuolar Myopathies, and speculated that they may be myofibers caught in the act of dying. Since these myofibers were identifiable by Dystrophin Encircled Vacuoles and invaginations with Intracellular Localization we coined the acronym DEVILs and aimed to determine their frequency, pathogenesis and correlation with myofiber loss. In whole transverse sections of young (1-6 month) and elderly (22-26 month) C57Bl/6j mouse muscles, DEVILated myofiber number correlated with myofiber loss, being increasingly prevalent in aged extensor digitorum longus (R = 0.7, p < 0.001) and soleus (R = 0.6, p = 0.004) muscles, whilst rare in myofiber loss resistant muscles (cleido- and sternomastoid). In a cell viability dye-exclusion test, 17 ± 14% of DEVILated myofibers stained positive and were accompanied by immunoglobulin infiltration compared to 1 ± 1% of normal myofibers (p = 0.029). Virtually all DEVILs were acid-phosphatase reactive but contained p62 immunoreactivity and periodic acid-Schiff stained plaques. Compared to normal myofibers, BNIP3 immunostaining in DEVILated myofibers was reduced, whilst MAP-LC3b was indifferent. Cleaved-caspase 3 immunoreactivity was marginally elevated in DEVILated myofibers, but unaccompanied by nuclear DNA fragmentation. DEVILated myofibers were also identified in elderly rat (24 month) and cadaveric human (78 years) muscles. We argue that DEVIL formation reflects a previously undescribed fibre death process via a mechanism involving autophagic dysfunction and that the process may represent our first direct insight into the mechanism by which myofibers are lost in old age.

Modelling dichotomously marked muscle fibre configurations.

Human skeletal muscle consists of contractile elements (fibres) that may be differentiated according to their physiological and biochemical properties. The different types of fibre are distributed throughout each muscle, with the pattern (when viewed as a cross-section) of cell distribution being an important determinant of the functional properties of each muscle. It is well known that the proportions and distributions of muscle fibre types change with advancing age or disease, but few studies have quantitatively investigated these changes. A better knowledge of the nature of changes in muscle fibre distributions is an essential requirement for future development of therapies and interventions directed at maintaining or restoring good muscle function. In this work, we examine several statistical methods designed to gauge the departure of a dichotomously labelled muscle fibre distribution from that of a random fibre-type dispersal. These methods are also applicable to a wide range of biological investigations in which the spatial distribution of cells or specimens underpins an important biological principle. This work includes the proposal of a novel technique, based on weighted kernel-smoothed density ratios, which can account for the variable areas of the individual fibres. We illustrated the methodology by using a number of real-data examples, and we employed a comprehensive set of simulations to assess the empirical power and false-positive rates of these tests.

Age-related loss of muscle fibres is highly variable amongst mouse skeletal muscles.

Sarcopenia is the age-related loss of skeletal muscle mass and strength, attributable in part to muscle fibre loss. We are currently unable to prevent fibre loss because we do not know what causes it. To provide a platform from which to better understand the causes of muscle fibre death we have quantified fibre loss in several muscles of aged C57Bl/6J mice. Comparison of muscle fibre numbers on dystrophin-immunostained transverse tissue sections at 6 months of age with those at 24 months shows a significant fibre loss in extensor digitorum longus and soleus, but not in sternomastoid or cleidomastoid muscles. The muscles of the elderly mice were mostly lighter than their younger counterparts, but fibres in the elderly muscles were of about the same cross-sectional area. This study shows that the contribution of fibre death to sarcopenia is highly variable and that there is no consistent pattern of age-related fibre loss between skeletal muscles.

Investigation of neuromuscular abnormalities in neurotrophin-3-deficient mice.

Neurotrophin-3 (NT-3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT-3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency-induced reduction in NT-3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1-43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve-mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1-43 were impaired in mature NT-3-deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype-dependent variation in the number of synaptic vesicles near the active zone. NT-3(+/-) mice had normal soleus muscle fiber numbers but their fibers had smaller cross-sectional areas and were more densely-packed than wild-type littermates. Moreover, the muscles of adult NT-3-deficient animals were weaker than those of wild-type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT-3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.

Developmental loss of NT-3 in vivo results in reduced levels of myelin-specific proteins, a reduced extent of myelination and increased apoptosis of Schwann cells.

This work investigates the role of NT-3 in peripheral myelination. Recent articles, based in vitro, propose that NT-3 acting through its high-affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT-3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P(0), and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT-3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT-3 as a major support factor for Schwann cells (Meier et al., (1999) J Neurosci 19:3847-3859) over-rides its potential role as an myelin inhibitor, with the net effect that loss of NT-3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT-3 null mutants showed increased expression of activated caspase-3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT-3 mutant animals suggesting that loss of NT-3 and resulting reduction in MAG levels may alter signaling at the axon-glial interface.

Nerve growth factor mRNA expression in the regenerating antler tip of red deer (Cervus elaphus).

Deer antlers are the only mammalian organs that can fully regenerate each year. During their growth phase, antlers of red deer extend at a rate of approximately 10 mm/day, a growth rate matched by the antler nerves. It was demonstrated in a previous study that extracts from deer velvet antler can promote neurite outgrowth from neural explants, suggesting a possible role for Nerve Growth Factor (NGF) in antler innervation. Here we showed using the techniques of Northern blot analysis, denervation, immunohistochemistry and in situ hybridization that NGF mRNA was expressed in the regenerating antler, principally in the smooth muscle of the arteries and arterioles of the growing antler tip. Regenerating axons followed the route of the major blood vessels, located at the interface between the dermis and the reserve mesenchyme of the antler. Denervation experiments suggested a causal relationship exists between NGF mRNA expression in arterial smooth muscle and sensory axons in the antler tip. We hypothesize that NGF expressed in the smooth muscle of the arteries and arterioles promotes and maintains antler angiogenesis and this role positions NGF ahead of axons during antler growth. As a result, NGF can serve a second role, attracting sensory axons into the antler, and thus it can provide a guidance cue to define the nerve track. This would explain the phenomenon whereby re-innervation of the regenerating antler follows vascular ingrowth. The annual growth of deer antler presents a unique opportunity to better understand the factors involved in rapid nerve regeneration.

Y-box factor YB1 controls p53 apoptotic function.

Nuclear localization and high levels of the Y-box-binding protein YB1 appear to be important indicators of drug resistance and tumor prognosis. YB1 also interacts with the p53 tumor suppressor protein. In this paper, we have continued to explore YB1/p53 interactions. We report that transcriptionally active p53 is required for nuclear localization of YB1. We go on to show that nuclear YB1 regulates p53 function. Our data demonstrate that YB1 inhibits the ability of p53 to cause cell death and to transactivate cell death genes, but does not interfere with the ability of p53 to transactivate the CDKN1A gene, encoding the kinase p21(WAF1/CIP1) required for cell cycle arrest, nor the MDM2 gene. We also show that nuclear YB1 is associated with a failure to increase the level of the Bax protein in normal mammary epithelial cells after stress activation of p53. Together these data suggest that (nuclear) YB1 selectively alters p53 activity, which may in part provide an explanation for the correlation of nuclear YB1 with drug resistance and poor tumor prognosis.

Change in mitochondrial membrane potential is the key mechanism in early warm hepatic ischemia-reperfusion injury.

The mitochondrion has been proposed to be both a target and a perpetuator of hepatic ischemia-reperfusion (IR) injury because of its reactive oxygen species (ROS) formation. Our hypothesis is that subcellular derangement in mitochondrial function is one of the earliest steps leading to the early IR-mediated loss of hepatocellular integrity. Under chloralhydrate anesthesia (36 mg/kg BW), Sprague-Dawley rats (n=7) were subjected to 40 min of warm hepatic lobular ischemia followed by 60 min reperfusion. Rats (n=7) without hepatic IR were used as controls. The fluorochromes rhodamine 123 and bisbenzimide were administered intravenously for observation of changes in mitochondrial membrane potential and hepatocellular viability, respectively. Intravital fluorescence microscopy (IVFM) was performed prior to ischemia and at 15, 45, and 60 min after reperfusion in the experimental group and at corresponding time points in the control group. A parallel relationship between mitochondrial membrane potential and cell viability as reflected in a concomitant reduction in nuclear and cytoplasmic fluorescence intensity during IR was demonstrated (r2=0.76, P<0.05). The diminution in fluorescence intensities also correlated significantly with the elevation in plasma transaminase activities (r2>0.90, P<0.05). Our data suggested that alteration in mitochondrial membrane potential is a critical subcellular event leading to hepatocellular damage in the early phase of hepatic IR injury.

Neurotrophin-3 null mutant mice display a postnatal motor neuropathy.

This paper examines early postnatal development of the neuromuscular system in mice with a null mutation in the gene for neurotrophin-3. We report that alpha-motoneurons at first develop substantially normally, despite a known 15% deficit in their somal size [Woolley et al. (1999)Neurosci. Lett., 272, 107-110.] and the absence of proprioceptive input [Ernfors et al. (1994)Cell, 77, 503-512]. At birth, motor axons have extended into the muscle, forming normal-looking neuromuscular junctions with focal accumulations of acetylcholine receptors. Detailed ultrastructural analysis does however, reveal subtle abnormalities at this time, particularly a decrease in the extent of occupancy of the postsynaptic site by nerve terminals, and a small but significant deficit in myofibre number. After the relative normality of this early neuromuscular development, there then occurs a catastrophic postnatal loss of motor nerve terminals, resulting in complete denervation of hindlimb muscles by P7. In systematic semi-serial samples through the entire muscle endplate zones, no neuromuscular junctions can be found. Intramuscular axons are fragmented, as shown by both electron microscopic observations and neurofilament immunohistochemistry, and alpha-bungarotoxin detection of acetylcholine receptors indicates dispersal of the junctional accumulation. At earlier times (postnatal days three and four) the terminal Schwann cells show ultrastructural abnormalities, and preliminary observations suggest marked disturbance of myelination. Based on comparison with other literature, the peripheral nerve degeneration seems unlikely to have arisen as a secondary effect of de-afferentation. We discuss whether the neural degeneration is secondary to the disturbance of Schwann cell function, or due directly to a loss of neurotrophin-3 based support of the motoneuron.

Development of a mammalian series-fibered muscle.

This study examines the processes by which multiply innervated, serially fibered mammalian muscles are constructed during development. We previously reported that primary myotubes of such a muscle, the guinea pig sternomastoid muscle, span from tendon to tendon and are innervated at each of the muscle's four innervation zones. Secondary myotubes form later, in association with each point of innervation (Duxson and Sheard, Dev. Dyn., 1995; 204:391-405). We now describe the further growth and development of the muscle. Secondary myotubes initially insert onto and grow along the primary myotube. However, as they reach a critical length, they encounter other secondary myotubes growing from serially adjacent innervation zones and may transfer their attachment(s) to these serially positioned secondary myotubes. Other secondary myotubes maintain attachment at one or both ends to their primary myotube. Thus, an interconnected network of primary and secondary myotubes is formed. Patterns of reactivity for cell adhesion molecules suggest that early attachment points between myotubes are the embryonic precursors of adult myomyonal junctions, characterized by the expression of alpha7Bbeta1 integrin. Finally, the results show that secondary myotubes positioned near a tendon are generally longer than those lying in the mid belly of the muscle, and we suggest that the environment surrounding the tendinous zone may somehow stimulate myotube growth.

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.