Involvement of the muscle-tendon junction in skeletal muscle atrophy: an ultrastructural study.

BACKGROUND: The muscle-tendon junction (MTJ) is a physiologically vital tissue interface and a highly specialized region in the muscle-tendon unit. It is the weakest point in the muscle-tendon unit, making it susceptible to strain injuries. Nonetheless, knowledge of the pathological changes affecting this region and of its response to the atrophy process is very limited. The aim of the study was to examine MTJ ultrastructural morphology in patients with different conditions that induce skeletal muscle atrophy and to attempt a grading of the atrophy process. MATERIALS AND METHODS: Fifteen patients undergoing amputation in the distal or proximal third of the lower leg due to chronic or acute conditions were divided into two groups. Specimens of gastrocnemius muscle collected at the time of surgery were analyzed by histology and electron microscopy. The contact between muscle and tendon was measured using a dedicated software that calculated semi-automatically the base (B) and perimeter (P) of muscle cell finger-like processes at the MTJ. RESULTS: Electron microscopy. The cells in the atrophic muscle of the chronic group were shallow and bulky. In the acute group, the myotendinous endings differed significantly in their structure from those of the chronic group. In atrophic muscle, the contact between muscle and tendon was reduced by quantitative and qualitative changes in the myotendinous endings. The B/P ratio allowed definition of three grades of myotendinous ending degeneration. DISCUSSION: It is unclear whether degenerative changes induced by immobilization in muscle and, specifically, the MTJ are temporary and reversible or permanent. CONCLUSIONS: This preliminary study suggested a classification of ultrastructural MTJ changes into grade 0, reflecting a quite normal MTJ; grade 1, an intermediate process that might lead to irreversible atrophy or to recovery, spontaneously or with drug therapy; and grade 2, irreversible process with complete structural alteration.

Decreased numeric density of succinic dehydrogenase-positive mitochondria in CA1 pyramidal neurons of 3xTg-AD mice.

Alzheimer disease (AD) is associated with mitochondrial dysfunction. In this study, we investigated succinic dehydrogenase (SDH) activity in mitochondria of hippocampal CA1 pyramidal neurons obtained from 10-month-old 3xTg-AD mice, an animal model of AD, as well as from age-matched control mice PS1-KI. In SDH-positive mitochondria, we measured numeric density (Nv, number of mitochondria/microm(3) of cytoplasm), average organelle volume (V), volume density (Vv, volume fraction of mitochondria/microm(3) of cytoplasm), average length (Fmax), and the ratio (R) between the total area of the cytochemical precipitate due to SDH activity and the total mitochondrial area. Our results indicate that 3xTg-AD mitochondria show a significant decrease of Nv, increase in V and Fmax, as well as a trend toward a reduction of R, whereas Vv is unchanged. Our findings further support the idea that mitochondrial dysfunction is involved in AD and are in line with studies indicating that both amyloid precursor protein (APP) and amyloid-beta (Abeta) localize to mitochondria.

Structural synaptic remodeling in the perirhinal cortex of adult and old rats following object-recognition visual training.

The ultrastructural features of layer II synapses in the perirhinal cortex of adult (4- to 6-month-old) and old (25- to 27-month-old) rats exposed to a six-session object recognition visual training were investigated by morphometric methods. The comparative analysis showed a higher synaptic numeric density, a lower synaptic average area, and a lower percentage of megasynapses (S > 0.5 microm2) in old trained rats versus controls, and a higher percentage of small (S < 0.15 microm2) junctions in adult trained rats versus controls. The more marked synaptic remodeling underlying memory consolidation in the perirhinal cortex of old rats might reflect a pre-existing lower dynamic status.

Ethanol-induced decrease of the expression of glucose transport protein (Glut3) in the central nervous system as a predisposing condition to apoptosis: the effect of age.

We measured the effect of chronic ethanol administration on the expression of Glut3 in the cerebellum and hippocampus of adult and old rats. Glut3 expression significantly decreased in aging, in ethanol-treated rats vs. age-matched controls, and in adult- vs. old ethanol-treated rats. These findings lend consistent support to the hypothesis that disturbances of glucose metabolism due to ethanol may constitute an unfavorable condition predisposing to neuronal death.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

Age-related effects of moderate alcohol consumption on GAP-43 levels in rat hippocampus.

The effects of moderate intake of ethanol and ageing were investigated on the levels of the growth-associated protein GAP-43, whose expression has been used as an indicator of axonal growth during development, regeneration and remodelling of synaptic connections. Groups of female Wistar rats (12 and 24 months of age), were alcohol-fed for one month while age-matched control groups received an isocaloric diet. A quantitative evaluation of GAP-43 was performed in hippocampus and in hippocampal selected areas in view of the vulnerability of this complex to alcohol aggression by means of two different methods, namely Western blot analysis and immunohistochemistry. While the former measures total extractable GAP-43, the latter allows visualisation of in situ changes in topographical distribution of GAP-43. Western blot analysis revealed an age-dependent reduction (-47%) and an ethanol-associated increase (81%) of GAP-43 demonstrated only in the old group. Conversely, quantitative immunohistochemistry of GAP-43 in the entire hippocampus showed a non-significant ethanol-related decrement in 24-month-old rats (-30%), although the age-dependent reduction was confirmed. Ageing was associated with a decrement of GAP-43 immunostaining in CA3 stratum radiatum (CA3) and in inner molecular layer of dentate gyrus (IML). Treatment determined a decrease of GAP-43 immunostaining in adult rat CA3 and IML and no change in CA1 stratum radiatum (CA1). Our results suggest that immunohistochemistry evaluation underestimates GAP-43 levels in ethanol-treated animals possibly as a consequence of conformational changes induced by alcohol, resulting in non-targeting of the specific antibody. Western blot analysis demonstrate that although there is a reduction of GAP-43 levels in hippocampus of aged rats, this structure retain a remarkable potential to compensate for ethanol toxicity during ageing.

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.

Mapping of mitochondrial metabolic competence by cytochrome oxidase and succinic dehydrogenase cytochemistry.

To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/microm(3) of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:1191-1192, 2001)

Cellular distribution of GAP-43 mRNA in hippocampus and cerebellum of adult rat brain by in situ RT-PCR.

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:1195-1196, 2001)

Zinc-bound metallothioneins as potential biological markers of ageing.

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

Distribution of map2 in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry.

The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging.

Quantitative immunohistochemistry of glucose transport protein (Glut3) expression in the rat hippocampus during aging.

Immunohistochemistry of Glut3 (45 kD), an integral membrane peptide mediating the transport of glucose in neurons, was carried out in the hippocampus of 3- and 28-month-old rats to assess the effect of age on energy metabolism. Free-floating sections of fixed-frozen hippocampi were processed for quantitative immunohistochemistry of Glut3. A rabbit affinity-purified antibody identified Glut3 immunoreactivity. Glut3 staining was intense in neuropil, axons, and dendrites, whereas nerve cell bodies were unstained. With aging, Glut3 reactivity was significantly decreased in the inner molecular layer of the hippocampal dentate gyrus (-46%) and the mossy fibers of the CA3 sector (-34%), whereas the stratum radiatum of CA1 did not show any difference due to age. These data document an age-dependent decrease in Glut3 expression in discrete areas of rat hippocampus. Glut3 constitutes the predominant glucose transporter in neurons and is found abundantly in regions with high synaptic density characterized by frequent bursts of function-adequate metabolic activity. Our findings therefore lend further support to the critical role of an impaired metabolism in age-related brain dysfunctions and disease.(J Histochem Cytochem 49:671-672, 2001)

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella.

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

Aging-like alterations of SDH activity in Purkinje cell mitochondria of adult vitamin-E deficient rats.

The ultrastructural features of perikaryal mitochondria positive to the copper ferrocyanide cytochemical reaction due to SDH activity were investigated in Purkinje cells of adult rats fed a vitamin E (α-tocopherol) deficient diet (AVED) for 11 months. The mitochondrial volume fraction (volume density: Vv), the number of organelles/µm(3) of tissue (numeric density: Nv) and their average volume (V) were estimated by computer-assisted morphometry. The data obtained were compared with our previous results on 3, 12 and 24 month-old normally fed rats. In a comparison with age-matched controls, AVED animals showed significant decreases of the three morphometric parameters taken into account. These reductions were also observed in old, normally fed rats vs. the young and adult groups, but in AVED rats Vv and V decreased to a higher extent. In adult control animals, the percent of larger organelles (0.32 µm(3) >) decreases to less than 1%. Vitamin E deficiency resulted in a steeper reduction of this fraction of organelles, i.e. only 0.5% in the 0.24-0.32 µm(3) size range accounted for the largest mitochondria in the AVED group. Taken together, these data document a significant impairment of mitochondrial efficiency in old and AVED rats. We interpret these findings to support that the underlying processes of aging and vitamin E deficiency may share common mechanisms. Considering the antioxidant action of α-tocopherol and the SDH role in cellular bioenergetics, inadequate protection from free radical attacks appears to represent an important determinant in the age-related decline of the mitochondrial metabolic competence.

Muscle involvement in rheumatoid arthritis: an ultrastructural study.

An electron microscopic investigation has been carried out on muscle bioptic samples from patients affected by rheumatoid arthritis (RA). This study was undertaken to seek further ultrastructural alterations affecting striated muscles in RA pathology. Bioptic samples were collected on a total of 30 surgical interventions of hip (10), knee (8), and foot (12). This yielded three muscle types: gluteus maximus, vastus lateralis, and extensor digitorum communis. Muscle samples from 12 patients with no RA stigmata, selected to match RA patients by age and gender, constituted the control group. Tissue samples were prepared both for conventional histochemical methods and according to conventional electron microscopic procedures, including morphometric analysis. Although to a different extent in each sample, in muscles from RA vs. controls the authors observed the simultaneous presence of discrete muscular alterations such as wider separation of myofibrils, myelin figures, dilated sarcotubular system, pleomorphic mitochondria, myofibril flaking, and lipofuscin deposition in the subsarcolemmal region. In addition to a progressive atrophy, the above findings are suggestive of rheumatoid myositis and lend further support to the still poorly documented presence of an idiopathic inflammatory myopathy and inclusion body myositis associated with RA.

Causes and consequences of damage to mitochondria : morphological aspects.

In recent years the role played by mitochondria in cellular aging has become the focus of intensive research. The concept that these energy-producing organelles are involved in aging derives from the views of Harman (1) and Gerschman (2) linking senescence to the damaging effects of free radicals, especially those released in the mitochondrial respiratory chain.