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.

Morphometry of axon cytoskeleton at internodal regions of rat sciatic nerve during aging.

BACKGROUND: Nerve endings undergo a lifespan morphofunctional modulation which is reported to be markedly impaired with aging. Neurone structural remodelling is in charge of processes occurring in the nerve cell soma, however the axonal transportation of organelles and molecules by cytoskeletal elements plays a very important role in the morphological rearrangements taking place at peripheral compartments. OBJECTIVE: To assess the involvement of axonal ultrastructure in the reported age-related decline of slow axoplasm flow mechanisms, we carried out a morphometric study of axon cytoskeleton in aging. METHODS: Female Wistar rats (3, 12 and 30 months of age) were anesthetized and perfused with saline followed by a fixation solution (glutaraldehyde 5% + formalin 2% in 0.1 cacodylate buffer pH 7.4). The excised sciatic nerves were processed according to conventional electron microsopic procedures. Axons sectioned perpendicularly to their longitudinal axis at the internodal region (mean axoplasm area: 18.25-26.5 microm(2)) were sampled by a systematic random procedure. The overall number of neurofilaments (No.Nfs) and microtubules (No.Mts) per total axoplasm area analysed, the numeric density (number/microm(2) of axoplasm area) of neurofilaments (NaNfs) and microtubules (NaMts), the myelin thickness, the number of myelin lamellae and the R proportion [No. Nfs/(No.Nfs + No.Mts)] were the parameters measured by computer-assisted semiautomatic methods. RESULTS: No.Nfs, NaNfs, myelin thickness and the number of myelin lamellae did not change between 12 and 30 months of age, while a significant increase of these parameters was found in a comparison with younger rats. No.Mts and NaMts were significantly increased at 12 vs. 3 as well as at 30 vs. 12 months of age, respectively. R proportion did not show any difference due to age. CONCLUSIONS: The present findings support that the dynamic condition of the axonal cytoskeleton appears to be preserved at a high extent in aging. Thus, the intra-axonal defective spacing of cytoskeletal elements (e.g. neurofilaments), rather than their number, is proposed to contribute to the decline of the slow axonal transport of organelles and molecules reported in aging.

Quantitation of intraplatelet Ca++ deposits as a potential marker of senility.

OBJECTIVE: To perform a morphometric evaluation of calcium deposits in human platelets as a quantitative procedure to seek a potential marker of senility in a peripheral cellular model. STUDY DESIGN: In human blood samples from middle-aged, healthy volunteers, the intraplatelet calcium content was cytochemically evidenced by the oxalatepyroantimonate (OPA) reaction. The number and area of OPA aggregates per square micrometer of total sampled area, the area of the deposits per square micrometer of platelet surface and the percentage of positive platelets were the ultrastructural features calculated by computer-assisted image analysis. RESULTS: OPA precipitates were easily identified in all the samples evaluated. The area of OPA deposits per square micrometer of platelet surface was rather constant not only among the measurements performed on the same sample but also comparing the different subjects analyzed. Other OPA deposit features showed higher variabilities; thus, to obtain a representative sample from each patient, several measurements had to be carried out. CONCLUSION: Quantitation of calcium deposits may be of help in evidencing increased Ca++ sequestering activity by platelets, supposedly due to altered calcium homeostasis. The OPA cytochemical procedure visualizes millimolar quantities of Ca++ ions; thus, only high calcium concentration sites (granules) can be detected by morphometric methods.

Dietary restriction modulates synaptic structural dynamics in the aging hippocampus.

A computer-assisted morphometric study has been carried out on the synaptic ultrastructural features in the hippocampus of 14-month old (DR14) and 27-month old (DR27) dietary restricted (-50% lipids and -35% carbohydrates) rats. Age-matched controls were maintained on an ad libitum (AL) feeding schedule. Synaptic numeric density (Nv), surface density (Sv) and average area (S) were the parameters measured. In old AL vs. adult AL animals, Nv decreased to a not significant extent, while S increased and Sv decreased significantly. In DR14 rats vs. AL littermates Nv increased significantly, but S and Sv were unchanged. DR27 rats vs. age-matched AL controls showed a significant increase of Nv and Sv while S was significantly decreased. Comparing DR14 vs. DR27, no significant difference due to age was documented. Both in DR14 and in DR27 groups the percent distribution of S showed a marked increase of smaller contact zones. Despite reporting on discrete aspects of synaptic ultrastructure, Nv and S are supported to be in an inverse relationship which aims at maintaining Sv constant. Thus, these three ultrastructural parameters when taken together per experimental group, appear to provide information on synaptic morphological rearrangements. In this context, the percent increase of smaller synapses in DR animals is consistent with the idea of a marked remodelling process. Considering previous data from the same groups of rats reporting significant changes in neuronal membrane lipid composition and fluidity, we interpret our findings to account for a positive modulation of dietary restriction on the synaptic structural dynamics.

Effect of age on synaptic size in human brain tissue proximal to tumor masses.

OBJECTIVE: To measure the effect of age on synaptic morphologic rearrangements in human brain tissue peripheral to space-occupying lesions. STUDY DESIGN: Synaptic length (L) was measured interactively by computer-assisted image analysis in brain tissue samples from adult (mean age, 39 years) and old (mean age, 67.2 years) patients. Each group consisted of five subjects. RESULTS: L was reduced by 9% in the old vs. adult group of patients. A percent distribution of the data showed that junctional areas smaller than 0.25 micron accounted for 47.2% in old patients and 31% in the adults. CONCLUSION: The present findings are in contrast with current literature data documenting a significant increase in the percent of enlarged contact zones in the senile brain. We interpret these results in terms of synaptic dynamic morphology and suggest that they may reflect alterations of neuronal adaptive capabilities due both to the pathologic condition and age of the patients.

Quantitative cytochemistry of succinic dehydrogenase activity in rat mitochondria.

OBJECTIVE: To set up a computer-assisted morphometric procedure to cytochemically measure the activity of succinic dehydrogenase (SDH). STUDY DESIGN: In rat mitochondria from Purkinje cell perikarya and frozen muscle samples, SDH activity was selectively evidenced by the copper ferrocyanide method. On the SDH-positive organelles we measured the following parameters: number of mitochondria per cubic micrometer (numeric density [Nv]), volume fraction of organelles per cubic micrometer (volume density [Vv]), average mitochondrial volume (V) and intramitochondrial area density of the SDH reaction (area of the precipitates/total mitochondrial area [R]). RESULTS: In both fresh Purkinje cells and frozen muscle cells the positive organelles were sharply evident. By considering Vv, Nv and V altogether in a given experimental group, a reliable evaluation of the morphologic rearrangements of the cellular metabolic hardware can be obtained. Measurements of R provides information on the functional efficiency of each organelle. CONCLUSION: Quantitation of SDH-positive mitochondria is closely associated with the amount of enzyme molecules present and active within the organelles. Thus, quantitative assessment of the copper ferrocyanide reaction contributes to the evaluation of mitochondrial metabolic competence.

Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging.

The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

Age-dependent decrease in the activity of succinic dehydrogenase in rat CA1 pyramidal cells: a quantitative cytochemical study.

A computer-assisted morphometric study has been carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria positive to the copper ferricyanide cytochemical reaction for succinic dehydrogenase (SDH) in rats of 3, 12 and 23 months of age. The cytoplasmic volume fraction occupied by the positive mitochondria (Volume density: Vv), the number of organelles/micron 3 of CA1 pyramidal cell cytoplasm (Numerical density: Nv) and the average mitochondrial volume (V) were automatically calculated by means of computer-assisted morphometry. Vv was significantly decreased in 23-month-old animals versus the other age groups. Nv was unchanged between 3 and 12 months of age, but was decreased to a significant extent in old animals. V did not undergo significant changes in the three age groups taken into account. In the old animals the percent of organelles smaller than 0.16 micron 3 is above 20%, while in the young and adult groups the same size of mitochondria accounts for 7 and 3%, respectively. Thus, a reduction in the number of medium sized organelles appears to be responsible for the decrease in Vv due to age. Since SDH activity is known to support maximum rates of respiration, quantitative estimation of the active mitochondria provides information on the metabolic competence of the cells investigated when energy demand is high. In this context, our present findings document that a significant impairment in the efficiency to match actual energy provisions occurs in old CA1 pyramidal cells.

Acetylcarnitine modulation of the morphology of rat hippocampal synapses.

OBJECTIVE: To measure the effect of chronic administration of acetylcarnitine (ALCAR) on the morphologic plasticity of rat hippocampal synapses. STUDY DESIGN: Computer-assisted morphometry was carried out on the ultrastructure of hippocampal synapses in 6-, 12- and 22-month-old rats and in age-matched animals chronically treated with ALCAR from the age of 1 month up to when they were killed. Synaptic numeric (Nv) and surface (Sv) densities as well as the synaptic average area (S) were measured in the dentate gyrus supragranular layer. RESULTS: In control animals, Nv was constant between 6 and 12 months of age but significantly decreased in 22-month-old rats, S did not show significant differences due to age and Sv was unchanged between 6 and 12 months but decreased significantly in the old animals. In ALCAR-treated rats, Nv increased and S decreased significantly vs. age-matched controls. Sv showed lifespan constancy among the age groups analyzed. In ALCAR-treated rats the number of synapses smaller than 0.08 microns 2 increased by 18%, 9% and 10% at 6, 12 and 22 months of age, respectively. CONCLUSION: We interpret these findings to represent positive modulation of the synaptic structural dynamics in ALCAR-treated animals through improvements in energy provision at nerve terminals.

Morphologic changes in cerebellar mitochondria during aging.

OBJECTIVE: To seek age-dependent morphofunctional changes in mitochondrial metabolic competence in the rat cerebellum. STUDY DESIGN: Three-, 12- and 24-month-old female Wistar rats were used for the present study. Each group consisted of five animals. The cytoplasmic fraction occupied by mitochondria (volume density, Vu), number of organelles per cubic micrometer (numerical density, Nu) and mitochondrial average volume (V) were measured in Purkinje cell perikaryal organelles histochemically stained to reveal succinic dehydrogenase activity in the three groups analyzed. RESULTS: Vu did not show significant differences due to age. Nu was constant between 3 and 12 months of age but decreased significantly in old rats. V did not show significant age-dependent differences. CONCLUSION: The present findings further extend the results of our previous studies on the ultrastructure of synaptic mitochondria and support the concept of marked metabolic impairment as a basal detrimental condition in the process of brain aging. As a marker of metabolically active mitochondria at high rates of cellular respiration, our present data document that with advancing age the number of efficient organelles decreases and that this may be of critical importance for the proper function of nerve cells when the energy demand is high.

Deterioration threshold of synaptic morphology in aging and senile dementia of Alzheimer's type.

OBJECTIVE: To search for a deterioration threshold of synaptic ultrastructure in physiologic aging and senile dementia of Alzheimer's type (SDAT). STUDY DESIGN: The numerical density (Nu), average area (S) and surface density (Su) of the synaptic contact zones were investigated in the hippocampus and cerebellum from adults, elderly and SDAT patients by means of computer-assisted morphometry. RESULTS: The Nu and surface Su densities of the synapses decreased while the size of S enlarged in elderly and SDAT patients vs. the adults. By plotting Nu vs. S, the adult group showed a very high percentage of small junctions. In contrast, elderly and dementia patients demonstrated two overlapping populations of enlarged contacts. In SDAT the synapse-to-neuron ratio was decreased by 48% in the hippocampus and by 56% in the cerebellum. CONCLUSION: Our findings support the concept that the degeneration of synaptic contacts per se should be considered a crucial step in the progression of senile dementia, but the identification of a discrete deterioration threshold of synaptic morphology between aging and SDAT is not feasible at present.

Synaptic structural dynamics and aging.

Synaptic junctional areas are not immutable structures, on the contrary, they are remodelled throughout the individual's lifespan as a consequence of environmental stimulations. This adaptive capacity of the synapses is discussed from a morphological standpoint with reference to aging. In old subjects, the number of contacts and the total surface area of synaptic appositions per unit volume of tissue decrease significantly, while the average synaptic size increases at a different extent according to the CNS area taken into account. This increase in synaptic average area is due to a higher percent of a subpopulation of enlarged contacts supposed to represent either the degenerating junctional zones or a compensatory phenomenon counteracting the synaptic reduction in number. Recent studies on perforated synapses support that the enlarged junctions are possible intermediates in synaptic physiological restructuring, thus the higher percentage of this type of contacts in the old CNS may witness unaccomplished synaptic turnover cycles. Taking into account the high metabolic rate of nerve cells, an age-related impairment in energy provision at synaptic terminal regions may constitute an early and subtle alteration affecting synaptic dynamic morphology in aging.

The effect of vitamin E deficiency on the plasticity of cholinergic synapses: a computer-assisted morphometric study.

A computer-assisted morphometric study has been carried out on the ultrastructural features of the cholinergic synaptic junctional areas in the dentate gyrus supragranular layer of 11-month-old female Wistar rats and in littermates fed a vitamin E deficient diet for 10 months. The number of synapses/micro m(3) (numerical density: Nv), the average area of the junctional zones (S) and the total synaptic contact area/micro m(3) (surface density: Sv) were the three parameters taken into account. Nv and Sv significantly decreased, while S increased in the vitamin E deficient group. A size distribution of S showed that while in the normally fed animals the percentage of an enlarged synapses (0.16 micro m(2)>) accounts for 19% of the whole population, in the vitamin E deficient rats it raises at 44%. Relating the number of synapses to the number of dentate gyrus granule cells, the synapse-to-neurone ratio appeared to be decreased by 30% in the vitamin E deficient animals. It is currently reported that number (Nv) and size (S) of the synapses are in a close inverse relationship which aims at maintaining constant the total synaptic surface area (Sv) in a discrete volume of the neuropil, therefore, taken together per experimental group of rats, these parameters represent a reliable index of the synaptic morphological plasticity. Our present findings clearly document that the structural dynamics of the hippocampal cholinergic synapses are markedly affected by the absence of vitamin E from the diet and, in turn, support that an increased peroxidative stress may play a central role in the widely reported vulnerability of the cholinergic terminals with advancing age.

Vitamin E deficiency as a model of precocious brain aging: assessment by X-ray microanalysis and morphometry.

Vitamin E (alpha-tocopherol) is a known biological antioxidant able to quench the lipid peroxidation chain and to protect the cellular structures (e.g., plasma membranes) from the attack of free radicals which are reported to play a primary role in aging. To assess whether the absence of alpha-tocopherol from the diet of young laboratory animals may be considered a reliable model of precocious brain aging, intracellular ionic content of brain cortex pyramidal cells, ultrastructural features of synaptic contact zones, synaptic mitochondria and perykarial mitochondria positive to the succinic dehydrogenase (SDH) histochemical reaction with copper ferrocyanide have been investigated by X-ray microanalysis and computer-assisted morphometry in young, adult, old and 11-month-old vitamin E deficient rats. Our data document significant alterations of intracellular ionic content, synaptic contact areas and synaptic and perykarial mitochondria in aging. Vitamin E deficiency caused similar alterations in adult animals. Taking into account the known role of alpha-tocopherol in protecting the cellular membrane structure, we support that the common process underlying the changes found in aging and vitamin E deficiency is an excessive deterioration of the neuronal membrane.