Tiziana Casoli

One-month strawberry-rich anthocyanin supplementation ameliorates cardiovascular risk, oxidative stress markers and platelet activation in humans

Strawberries are an important fruit in the Mediterranean diet because of their high content of essential nutrients and beneficial phytochemicals, which seem to exert beneficial effects in human health. Healthy volunteers were supplemented daily with 500 g of strawberries for 1 month. Plasma lipid profile, circulating and cellular markers of antioxidant status, oxidative stress and platelet function were evaluated at baseline, after 30 days of strawberry consumption and 15 days after the end of the study. A high concentration of vitamin C and anthocyanins was found in the fruits. Strawberry consumption beneficially influenced the lipid profile by significantly reducing total cholesterol, low-density lipoprotein cholesterol and triglycerides levels (-8.78%, -13.72% and -20.80%, respectively; P<.05) compared with baseline period, while high-density lipoprotein cholesterol remained unchanged. Strawberry supplementation also significant decreased serum malondialdehyde, urinary 8-OHdG and isoprostanes levels (-31.40%, -29.67%, -27.90%, respectively; P<.05). All the parameters returned to baseline values after the washout period. A significant increase in plasma total antioxidant capacity measured by both ferric reducing ability of plasma and oxygen radical absorbance capacity assays and vitamin C levels (+24.97%, +41.18%, +41.36%, respectively; P<.05) was observed after strawberry consumption. Moreover, the spontaneous and oxidative hemolysis were significant reduced (-31.7% and -39.03%, respectively; P<.05), compared to the baseline point, which remained stable after the washout period. Finally, strawberry intake significant decrease (P<.05) the number of activated platelets, compared to both baseline and washout values. Strawberries consumption improves plasma lipids profile, biomarkers of antioxidant status, antihemolytic defenses and platelet function in healthy subjects, encouraging further evaluation on a population with higher cardiovascular disease risk.

Morphological adaptive response of the synaptic junctional zones in the human dentate gyrus during aging and Alzheimer's disease

A computer-assisted morphometric study has been carried out on ethanol phosphotungstic acid (E-PTA) stained synaptic junctions in the human dentate gyrus supragranular layer from adult, old and Alzheimers disease (AD)-affected patients. The number of synapses per unit volume of tissue (Nv = numerical density), the average area of the single junction (S) and the total area of the synaptic contact zones in a unit volume of tissue (Sv = surface density) were the 3 parameters taken into account. The synapse to neurone ratio was also calculated for each patient. During physiological aging, Nv and Sv significantly decreased and S increased, respectively. In the AD hippocampi, Nv and Sv underwent a further decrease which was in the range of more than 40% with reference to the adult values. S was the same as the old control group. In comparison with the adult values, the number of synapse/neurone decreased by 15.6 and 48% in old and AD patients, respectively. Nv, S and Sv, while reporting on discrete ultrastructural features of the synaptic junctional zones, are closely related to each other and, taken together per group of patients, may represent a reliable index of the morphological adaptive changes taking place at the synapses. Thus, the significant increase of S both in old and AD hippocampi may be regarded as a CNS plastic response to aging and disease, although the marked decrease of Nv and Sv supports that in AD synaptic ultrastructural alterations proceed beyond a critical threshold for functional recovery.

Morphological plasticity of synaptic mitochondria during aging

A morphometric investigation has been carried out on the synaptic mitochondria of cerebellar glomeruli in young, adult and old rats by means of a computer-assisted image analysis technique. Mitochondrial volume density (Vv), numerical density (Nv), average volume (V) and average length (Skeleton = Sk) were investigated in tissue samples fixed, embedded and sectioned according to conventional electron microscopic methods. Vv was unchanged in the three groups of age taken into account. Nv was significantly increased in adult vs. young animals, whereas it was decreased in the old group as compared to both the other two groups investigated. V and Sk showed the same age-dependent changes: they significantly decreased in the adult vs. the young and the old groups of rats while increased significantly in the old rats vs. both the adult and young animals. A percentage distribution of Sk demonstrated that in the old group 20.6% of the population of synaptic mitochondria accounts for elongated organelles (> 5 microns) as compared to 8.6% and 5.3% in young and adult animals, respectively. The present findings match the changes previously reported by us on the ultrastructure of synaptic contact zones both in rats and human beings, and support the idea of an age-dependent dynamic adaptation in the morphology of synaptic mitochondria to cope with the metabolic needs of the pattern of synaptic connectivity they subserve.

Release of beta-amyloid from high-density platelets: implications for Alzheimer's disease pathology.

Abstract:  The main component of Alzheimers disease (AD) senile plaques in the brain is amyloid‐β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Aβ and much evidence suggests that these cells may represent a useful tool to study both amyloidogenic and nonamyloidogenic pathways of APP processing. It has been demonstrated that platelets activated by physiological agonists, such as thrombin and collagen, specifically secrete Aβ ending at residue 40. To verify whether APP β‐processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Aβ released in the incubation media of 5 × 109 platelets/mL by enzyme‐linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Aβ40 levels were significantly higher in incubation media of 5 × 109/mL platelets in comparison with 108/mL platelets (normalized values), while Aβ42 levels were not affected by cell density. The ultrastructural analysis showed platelets at different phases of activation: some platelets were at earlier stage, characterized by granule swelling and dilution, others had granules concentrated in a compact mass in the cell centers within constricted rings of circumferential microtubules (later stage). Normally concentrated cells had the characteristic morphology of resting platelets. Our data suggest that high‐density platelets undergo activation likely by increased frequency of platelet–platelet collisions. This, in turn, determines the activation of APP β‐processing with consequent release of Aβ40. Investigating the biochemical pathways triggering Aβ secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.

Peripheral inflammatory biomarkers of Alzheimer’s disease: the role of platelets

Alzheimer’s disease is an age-dependent neurodegenerative disorder characterized by loss of neurons, synaptic degeneration, senile plaques and neurofibrillary tangles. Besides these hallmarks, increased accumulation of activated microglia, astrocytes and leukocytes adhering to postcapillary venules are observed in the affected brain areas, suggesting the presence of an ongoing inflammatory process. As neuroinflammation triggers the activation of peripheral immune system, many studies have analyzed circulating inflammatory biomarkers, including basal or stimulated levels of cytokines and related molecules in blood of Alzheimer’s patients, but with conflicting results. Platelets are an important source of amyloid-ß (Aß) in the circulatory system and play an important pro-inflammatory role. Upon activation, they adhere to leukocytes and endothelial cells by means of adhesive proteins like P-selectin, platelet endothelial cell adhesion molecule-1 (PECAM) and intercellular adhesion molecule-1 and -2 (ICAM-1 and -2) and secrete inflammatory mediators (chemokines, interleukins). In addition, platelets contain important enzymes involved in inflammatory intermediary synthesis like phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2), and recent reports demonstrated significant changes in platelet levels and activities in Alzheimer’s disease. Thus, as platelets represent an important link between Aß deposition and inflammatory reactions especially at endothelial level, they can be considered a valuable cellular model to evaluate potential peripheral inflammatory biomarkers in Alzheimer’s disease.

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. (J Histochem Cytochem 49:1065–1066, 2001)

Neuronal plasticity in aging: A quantitative immunohistochemical study of GAP-43 distribution in discrete regions of the rat brain

Age-related changes in neuroplasticity have been investigated considering the neuronal growth-associated protein GAP-43 as a marker of nerve cell structural adaptive capabilities. We carried out a quantitative immunohistochemical study on the distribution of GAP-43 in the molecular layer of the cerebellar cortex, in the inner molecular layer of the hippocampal dentate gyrus, in the stratum radiatum of the CA1 region, in layer 1 of the cingulate cortex and in the nerve fiber layer of the main olfactory bulb of 3-, 18- and 31-month-old Wistar rats. A decrease of GAP-43 immunoreactivity was observed in the old rats in comparison with the adult animals in all the 5 areas analyzed, although these variations were only statistically significant in the dentate gyrus, cingulate cortex and olfactory bulb. In these latter zones, GAP-43 immunolabeling is reduced by 54, 42 and 38%, respectively, in the old versus the adult group. Comparing these data with the age-dependent decrease of neuron density innervating the areas investigated, we support the hypothesis that the decline of GAP-43 observed in old animals documents a consistent reduction of axon plasticity in the inner molecular layer of the dentate gyrus and in layer 1 of the cingulate cortex. These results suggest an important role of GAP-43 as a marker of age-dependent deterioration of synaptic plasticity, especially in those areas of the brain involved in memory and emotional behavior.

A ketogenic diet increases succinic dehydrogenase (SDH) activity and recovers age-related decrease in numeric density of SDH-positive mitochondria in cerebellar Purkinje cells of late-adult rats

Ketogenic diets (KDs) have been applied in the therapy of paediatric epilepsy for nearly a century. Recently, beneficial results have also been reported on metabolic disorders and neurodegeneration, designating aged individuals as possible recipients. However, KDs efficacy decrease after the suckling period, and very little is known about their impact on the aging brain. In the present study, the effect on the neuronal energetic supply of a KD containing 20% of medium chain triglycerides (MCT) was investigated in Purkinje cells of the cerebellar vermis of late-adult (19-month-old) rats. The animals were fed with the KD for 8 weeks, and succinic dehydrogenase (SDH) activity was cytochemically determined. The following parameters of SDH-positive mitochondria were evaluated by the use of a computer-assisted image analysis system connected to a transmission electron microscope: numeric density (Nv), average volume (V), volume density (Vv), and cytochemical precipitate area/mitochondrial area (R). Young, age-matched, and old animals fed with a standard chow were used as controls. We found significantly higher Nv in MCT-KD-fed rats vs. all the control groups, in young vs. late-adult and old controls, and in late-adult vs. old controls. V and Vv showed no significant differences among the groups. R was significantly higher in MCT-KD-fed rats vs. all the control animals, and in old vs. young and late-adult controls. Present data indicate that the ketogenic treatment counteracted age-related decrease in numeric density of SDH-positive mitochondria, and enhanced their metabolic efficiency. Given the central role of mitochondrial impairment in age-related physio-pathological changes of the brain, these findings may represent a starting point to examine novel potentialities for KDs.

Synaptic and mitochondrial physiopathologic changes in the aging nervous system and the role of zinc ion homeostasis

Brain performances, e.g. learning and memory, decay during aging. Deterioration of synaptic junctions, as structural correlates of these key functions of the central nervous system, may play a central role in this impairment. Current research on the age-related changes of synapses is documenting that the numeric loss of contacts appears to trigger a compensatory reaction by the old CNS, i.e. the surviving junctional areas in old individuals are larger than in adult subjects. The final outcome of the balanced changes in synaptic number and size is that the overall synaptic junctional area per cubic micron of neuropil is also reduced in aging and this may account for the age-associated functional decay of CNS performances. Among the suggested determinants of synaptic deterioration in aging, a considerable number of recent studies support an early and pivotal role of the progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate. Quantitative ultrastructural studies together with cytochemistry of key enzymes of the respiratory chain (cytochrome oxidase and succinic dehydrogenase) have shown that mitochondrial dysfunctions play an early and central role in synaptic deterioration events associated with aging and neurodegenerative diseases. Among the various causes, the multiple mechanisms and molecules involved in zinc ion homeostasis have been supposed to be less efficient in the aging brain. Thus, a transient imbalance of free zinc ion concentration in the cytosol ([Zn2+]i) can be considered an unfavourable trigger of subtle mitochondrial damage and synaptic pathology.

Ketogenic Diets Cause Opposing Changes in Synaptic Morphology in CA1 Hippocampus and Dentate Gyrus of Late-Adult Rats

Ketogenic diets (KDs) have beneficial effects on several diseases, such as epilepsy, mitochondriopathies, cancer, and neurodegeneration. However, little is known about their effects on aging individuals. In the present study, late-adult (19-month-old) rats were fed for 8 weeks with two medium chain triglycerides (MCT)-KDs, and the following morphologic parameters reflecting synaptic plasticity were evaluated in stratum moleculare of hippocampal CA1 region (SM CA1) and outer molecular layer of hippocampal dentate gyrus (OML DG): average area (S), numeric density (Nv(s)), and surface density (Sv) of synapses, and average volume (V), numeric density (Nv(m)), and volume density (Vv) of synaptic mitochondria. In SM CA1, MCT-KDs induced the early appearance of the morphologic patterns typical of old animals (higher S and V, and lower Nv(s) and Nv(m)). On the contrary, in OML DG, Sv and Vv of MCT-KDs-fed rats were higher (as a result of higher Nv(s) and Nv(m)) versus controls; these modifications are known to improve synaptic function and metabolic supply. The opposite effects of MCT-KDs might reflect the different susceptibility to aging processes: OML DG is less vulnerable than SM CA1, and the reactivation of ketone bodies uptake and catabolism might occur more efficiently in this region, allowing the exploitation of their peculiar metabolic properties. Present findings provide the first evidence that MCT-KDs may cause opposite morphologic modifications, being potentially harmful for SM CA1 and potentially advantageous for OML DG. This implies risks but also promising potentialities for their therapeutic use during aging.