MRI characterization of rat brain aging at structural and functional level: Clues for translational applications.

Magnetic resonance imaging (MRI) paradigms, using non-invasive approaches, can provide relevant findings about brain aging. The attention has been primarily focused on neurodegenerative diseases, while little or nothing has been done to differentiate physiology from pathology. The present study aimed to test diffusion tensor imaging (DTI) and functional MRI (fMRI) metrics to analyze physiological age-related changes in rats at myelin structure and activation level; findings were validated by ex vivo histology. The purpose is to find comparable biomarkers in rodents and humans to allow a reliable translation from pre-clinical to clinical settings. Data evidenced: i) a significantly higher cerebrospinal fluid volume in middle-aged and aged vs. young rats; ii) a progressive alteration of white matter; iii) a significant reduction of evoked activity in aged animals. These results partially mirror the age-related changes in humans and may represent a preliminary step to find reliable tools for a lifelong monitoring with a value for the clinical practice (e.g., to provide support to the early diagnosis of dementia in asymptomatic subjects).

Testosterone administration increases synaptic density in the gyrus dentatus of old mice independently of physical exercise.

Testosterone and physical exercise administration have been shown to affect hippocampal morphology in adult rodents. In aged animals, similar data are only available after physical exercise. In this work we used ultrastructural quantitative morphometry to investigate the effect of testosterone administration on the hippocampal synapses of old mice, either alone or in combination with aerobic physical exercise. The inner molecular layer of the hippocampal dentate gyrus (IMLDG) and the molecular stratum of Ammon's horn 1 neurons (SMCA1) were investigated in 27-month-old male Balb/c mice randomly allocated to one of four experimental conditions (five mice each): sedentary control (C), testosterone administration (10 mg/kg once a week, TA), treadmill training (30 min a day, five days a week for 4 weeks at belt speed 8 m/min, 0% incline, TT) and testosterone administration plus treadmill training (TTTA). At the end of a four-week period, hippocampi were excised, fixed, and processed by ethanol phosphotungstic acid procedure to contrast synapses. The following variables were measured in electron micrographs: number of synapses/µm3 of tissue (Nv), total area of contact zones/µm3 of tissue (Sv), average area of the synaptic contact zone (S), and percentage of perforated synapses (%PS). ANOVA showed a statistically significant main effect of experimental condition for Nv and Sv in IMLDG, and for Sv in SMCA1 (p ≤ 0.003). The S and %PS were similar within group in ANOVA. Post-hoc analysis revealed a significant (p < 0.05) increase of Sv vs. C in SMCA1 and IMLDG after TT and TA, respectively. In IMLDG, Nv was significantly increased vs. C and TT after both TA and TTTA. Overall, results showed that testosterone increases synaptic density in IMLDG of old mice independently of physical exercise or changes in synaptic size. Instead, synaptic density in SMCA1 was only sensitive to physical exercise. These findings show that exogenous testosterone administration exerts a positive effect of on synapses in selected areas of the old mouse hippocampus.

Modulatory Effect of Aerobic Physical Activity on Synaptic Ultrastructure in the Old Mouse Hippocampus.

Aerobic physical exercise (APE) leads to improved brain functions. To better understand the beneficial effect of APE on the aging brain, a morphometric study was carried out of changes in hippocampal synapses of old (>27 months) Balb/c mice undergoing treadmill training (OTT) for 4 weeks in comparison with old sedentary (OS), middle-aged sedentary (MAS) and middle-aged treadmill training (MATT) mice. The inner molecular layer of the hippocampal dentate gyrus (IMLDG) and the molecular stratum of Ammon's horn1 neurons (SMCA1) were investigated. The number of synapses per cubic micron of tissue (numeric density, Nv), overall synaptic area per cubic micron of tissue (surface density, Sv), average area of synaptic contact zones (S), and frequency (%) of perforated synapses (PS) were measured in electron micrographs of ethanol-phosphotungstic acid (E-PTA) stained tissue. Data were analyzed with analysis of variance (ANOVA). In IMLDG, an effect of age was found for Nv and Sv, but not S and %PS. Similar results were found for exercise and the interaction of age and exercise. In post hoc analysis Nv was higher (60.6% to 75.1%; p < 0.001) in MATT vs. MAS, OS and OTT. Sv was higher (32.3% to 54.6%; p < 0.001) in MATT vs. MAS, OS and OTT. In SMCA1, age affected Nv, Sv and %PS, but not S. The effect of exercise was significant for Sv only. The interaction of age and exercise was significant for Nv, Sv and %PS. In post hoc analysis Nv was lower in OS vs. MAS, MATT and OTT (-26.1% to -32.1%; p < 0.038). MAS and OTT were similar. Sv was lower in OS vs. MAS, MATT and OTT (-23.4 to -30.3%, p < 0.004). MAS and OTT were similar. PS frequency was higher in OS vs. MAS, MATT and OTT (48.3% to +96.6%, p < 0.023). APE positively modulated synaptic structural dynamics in the aging hippocampus, possibly in a region-specific way. The APE-associated reduction in PS frequency in SMCA1 of old mice suggests that an increasing complement of PS is a compensatory phenomenon to maintain synaptic efficacy. In conclusion, the modulation of synaptic plasticity by APE gives quantitative support to the concept that APE protects from neurodegeneration and improves learning and memory in aging.

Oxidative Stress in Elderly with Different Cognitive Status: My Mind Project.

BACKGROUND: Biomarkers of oxidative stress have been associated with cognitive status in humans and have been proposed to guide prognosis/treatment in Alzheimer's disease (AD) and mild cognitive impairment (MCI). OBJECTIVE: The aim of this study was to compare oxidative stress status in the plasma of mild-moderate AD, MCI, and healthy elderly with normal cognition (HE) undergoing a non-pharmacological intervention including multi-modal cognitive training ("My Mind Project"). METHODS: A prospective randomized trial involving 321 elderly people enrolled in Marche Region, Italy. Each subject was randomly assigned to an experimental (cognitive training) or to a control group. Cognitive performances and biomarkers have been analyzed before intervention (baseline), immediately after termination (follow-up 1), after 6 months (follow-up 2), and after 2 years (follow-up 3). The biological antioxidant potential (BAP) to Diacron reactive oxygen metabolites (d-ROM) ratio has been used as an indicator of oxidative stress status and as outcome variable. RESULTS: We have found no differences in the oxidative status among AD, MCI, and HE. Neither did we find a significant effect of the intervention within experimental groups. Gender was the sole factor with a strong significant effect on BAP/d-ROM. CONCLUSIONS: Based on these results, the utility of biomarkers of oxidative stress to guide prognosis/treatment in AD or MCI seems to be limited by lack of specificity, large interindividual variability, and gender bias.

Potassium canrenoate compounding for administration via enteral feeding tubes: a physical and microbiological stability study.

BACKGROUND: Swallowing difficulties are arising in an increasing number of patients, especially in elderly people. When deglutition ability is completely compromised, enteral administration of a drug via feeding tubes is used. Licensed pharmacists have to compound the original solid forms to enable this drug therapy. OBJECTIVES: To evaluate the possibility of compounding original commercial tablets to produce a liquid formulation suitable for administering via a feeding tube. METHODS: Two liquid formulations containing potassium canrenoate 5 mg/mL were prepared: a standard solution obtained by solubilising raw material and an extemporaneous preparation obtained by dissolving film-coated 100 mg tablets. Spectrophotometric determinations (UV range) of the drug established chemical stability of the analyte up to 60 days. Samples were tested for microbial growth. Gravimetric quantifications of liquid formulations were used to check any weight loss during the different steps before enteral administration. RESULTS: UV data confirmed the chemical stability of potassium canrenoate up to 60 days. Samples showed no microbial growth. A higher weight loss was recorded in extemporaneous preparations than in the standard solution (10.7% vs 7.6%) according to the gravimetric quantification. CONCLUSION: It is possible to compound the original tablets into a liquid formulation suitable for administration via a feeding tube.

Effect of a Comprehensive Intervention on Plasma BDNF in Patients with Alzheimer's Disease.

A comprehensive intervention (CI) on patients with Alzheimer's disease was assessed by measuring plasmabrain-derived neurotrophic factor (pBDNF) and ADAS-Cog score (ADAS-Cogscore) before, immediately after (FU1), and 6 (FU2) and 24 months (FU3) after the CI. Baseline pBDNF was higher in patients with moderate AD (but not mild AD) than in healthy controls. At FU1, pBDNF and ADAS-Cogscore decreased significantly. At FU2 and FU3, patients' cognitive status worsened and pBDNF further increased versus baseline, suggesting that CI interruption may be a stress event that prevents return to homeostasis. CI exerted positive short-term effects, but more information is needed on long-term consequences.

Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment.

We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A.

Adapted physical exercise enhances activation and differentiation potential of satellite cells in the skeletal muscle of old mice.

During ageing, a progressive loss of skeletal muscle mass and a decrease in muscle strength and endurance take place, in the condition termed sarcopenia. The mechanisms of sarcopenia are complex and still unclear; however, it is known that muscle atrophy is associated with a decline in the number and/or efficiency of satellite cells, the main contributors to muscle regeneration. Physical exercise proved beneficial in sarcopenia; however, knowledge of the effect of adapted physical exercise on the myogenic properties of satellite cells in aged muscles is limited. In this study the amount and activation state of satellite cells as well as their proliferation and differentiation potential were assessed in situ by morphology, morphometry and immunocytochemistry at light and transmission electron microscopy on 28-month-old mice submitted to adapted aerobic physical exercise on a treadmill. Sedentary age-matched mice served as controls, and sedentary adult mice were used as a reference for an unperturbed control at an age when the capability of muscle regeneration is still high. The effect of physical exercise in aged muscles was further analysed by comparing the myogenic potential of satellite cells isolated from old running and old sedentary mice using an in vitro system that allows observation of the differentiation process under controlled experimental conditions. The results of this ex vivo and in vitro study demonstrated that adapted physical exercise increases the number and activation of satellite cells as well as their capability to differentiate into structurally and functionally correct myotubes (even though the age-related impairment in myotube formation is not fully reversed): this evidence further supports adapted physical exercise as a powerful, non-pharmacological approach to counteract sarcopenia and the age-related deterioration of satellite cell capabilities even at very advanced age.

Neonatal exposure to permethrin pesticide causes lifelong fear and spatial learning deficits and alters hippocampal morphology of synapses.

BACKGROUND: During the neurodevelopmental period, the brain is potentially more susceptible to environmental exposure to pollutants. The aim was to determine if neonatal exposure to permethrin (PERM) pesticide, at a low dosage that does not produce signs of obvious abnormalities, could represent a risk for the onset of diseases later in the life. METHODS: Neonatal rats (from postnatal day 6 to 21) were treated daily by gavage with a dose of PERM (34 mg/kg) close to the no-observed-adverse-effect level (NOAEL), and hippocampal morphology and function of synapses were investigated in adulthood. Fear conditioning, passive avoidance and Morris water maze tests were used to assess cognitive skills in rats, whereas electron microscopy analysis was used to investigate hippocampal morphological changes that occurred in adults. RESULTS: In both contextual and tone fear conditioning tests, PERM-treated rats showed a decreased freezing. In the passive avoidance test, the consolidation of the inhibitory avoidance was time-limited: the memory was not impaired for the first 24 h, whereas the information was not retained 72 h following training. The same trend was observed in the spatial reference memories acquired by Morris water maze. In PERM-treated rats, electron microscopy analysis revealed a decrease of synapses and surface densities in the stratum moleculare of CA1, in the inner molecular layer of the dentate gyrus and in the mossy fibers of the hippocampal areas together with a decrease of perforated synapses in the stratum moleculare of CA1 and in the inner molecular layer of the dentate gyrus. CONCLUSIONS: Early-life permethrin exposure imparts long-lasting consequences on the hippocampus such as impairment of long-term memory storage and synaptic morphology.

Effect of cognitive training on the expression of brain-derived neurotrophic factor in lymphocytes of mild cognitive impairment patients.

To identify biomarkers associated with cognitive stimulation of mild cognitive impairment (MCI) patients, we performed quantitative real-time reverse transcriptase polymerase chain reaction for brain-derived neurotrophic factor (BDNF) mRNA in peripheral lymphocytes of MCI and healthy subjects undergoing a multi-component cognitive training (CT) program. CT determined a significant decrease of BDNF mRNA levels in MCI (fold change=0.31) as compared to healthy subjects (fold change=0.86). It has been reported that in MCI there is an increase of BDNF serum levels, and our findings could indicate a positive effect of CT in restoring pre-disease levels of expression.

Contribution of non-reference alleles in mtDNA of Alzheimer's disease patients.

Many observations suggest that mutations of mitochondrial DNA (mtDNA) could be responsible for the neurodegenerative changes of Alzheimer's disease (AD). Here we examined the signal intensity of the four alleles of each mtDNA nucleotide position (np) in whole blood of AD patients and age-matched controls using MitoChip v2.0 array. Our analysis identified 270 significantly different nps which, with one exception, showed an increased contribution of non-reference alleles in AD patients. Principal component analysis (PCA) and cluster analysis showed that five of these nps could discriminate AD from control subjects with 80% of cases correctly classified. Our data support the hypothesis of mtDNA alterations as an important factor in the etiology of AD.

Early selective vulnerability of synapses and synaptic mitochondria in the hippocampal CA1 region of the Tg2576 mouse model of Alzheimer's disease.

Increasing experimental evidence indicates that synaptic alterations play a key role in cognitive decline in Alzheimer's disease (AD). Functional and structural synaptic changes progressively take place, beginning in the early phase of AD, mainly triggered by intracellular accumulation of soluble amyloid-ß (Aß) oligomers. These peptides also accumulate within mitochondria, heavily affecting their function and morphology, particularly in synaptic compartments. To better understand the role of mitochondrial impairment in synaptic alterations during the early stages of AD, a morphological investigation was performed by means of electron microscopy in the hippocampus of 3 month-old Tg2576 and transgene-negative littermate mice. In the stratum moleculare of CA1 pyramidal cells (SMCA1) of transgenic animals compared to controls, we found significantly larger and less numerous synapses, with a significantly reduced fraction of the perforated subtype, as well as significantly smaller and more numerous mitochondria. In contrast, no differences between the two groups of mice were found in the inner molecular layer of the dentate gyrus. The reduction of synaptic contacts in SMCA1 indicates a precocious vulnerability of this region, and the synaptic enlargement may reflect a compensating process aimed at maintaining the overall contact density. Accordingly, mitochondrial modifications may represent a plastic reactive phenomenon aimed at sustaining the increased energy needs for synaptic remodeling, since mitochondrial morphology was perfectly preserved and smaller mitochondria are metabolically more efficient. Thus, morphological changes occurring at synaptic level in SMCA1 of 3 month-old Tg2576 mice might reflect a precocious vulnerability associated with a residual plastic reactivity which may slow down functional alterations.

Platelets in Alzheimer's disease-associated cellular senescence and inflammation.

Alzheimer's disease (AD) is a complex degenerative disorder of the brain, associated with a progressive cognitive decline. Age is the main risk factor with almost half of the population above 90 years affected by this pathology. AD and brain aging share common molecular changes, so it has been hypothesized that AD could be a form of accelerated brain aging. In this context, senescenceassociated mechanisms could be a valuable target of investigation both to analyze the causes of this disease and to define therapeutic strategies. Senescent phenotypes of glia and neurons, as well as of peripheral cells, have been described in AD. Much evidence indicate that vascular impairment is a fundamental contributor to AD pathology and platelets are generally considered a key element because they represent the link between amyloid-ß (Aß) deposition, peripheral inflammation and endothelial senescence. Both activated and senescent platelets are a source of Aß, in addition activated platelets secrete many proinflammatory mediators that could contribute to increased peripheral inflammation and endothelial senescence. Treatments aimed to target peripheral endothelial senescence include antioxidants and some substances, such as aspirin, that modulate platelet aggregation and inflammatory response. Heparin has been proposed as a treatment for senile dementia and exhibits anti-inflammatory action as well as inhibitory effects on Aß assembly. Identifying peripheral targets for AD treatments could also result advantageous as it would be possible to monitor directly their efficacy. Nevertheless more research is needed to clarify all the different aspects and interactions of blood cells, vascular cells and their secretory products.

Early impairment of long-term depression in the perirhinal cortex of a mouse model of Alzheimer's disease.

Visual recognition memory is early impaired in Alzheimer's disease. Long-term depression of synaptic transmission in the perirhinal cortex is critically involved in this form of memory. We found that synaptic transmission was impaired in perirhinal cortex slices obtained from 3-month-old Tg2576 mice, and that 3,000 pulses at 5 Hz induced long-term depression in perirhinal cortex slices from age-matched control mice, but not in those from Tg2576 mice. To our knowledge, these data provide the first evidence of synaptic transmission and long-term depression impairment in the perirhinal cortex in an animal model of Alzheimer's disease, and the earliest synaptic deficit in Tg2576 mice.

Impairments of synaptic plasticity in aged animals and in animal models of Alzheimer's disease.

Aging is associated with a gradual decline in cognitive functions, and more dramatic cognitive impairments occur in patients affected by Alzheimer's disease (AD). Electrophysiological and molecular studies performed in aged animals and in animal models of AD have shown that cognitive decline is associated with significant modifications in synaptic plasticity (i.e., activity-dependent changes in synaptic strength) and have elucidated some of the cellular mechanisms underlying this process. Morphological studies have revealed a correlation between the quality of memory performance and the extent of structural changes of synaptic contacts occurring during memory consolidation. We briefly review recent experimental evidence here.

Dynamin binding protein gene expression and memory performance in aged rats.

Previous studies have shown that messenger RNA (mRNA) of the dynamin-binding protein (DNMBP), a scaffold protein regulating actin cytoskeleton and synaptic vesicle pools, is lower in neuropathologically-confirmed Alzheimer's brains. Here we investigated whether a deficit in long term memory formation during physiological aging is also associated with lower DNMBP expression. Hippocampal DNMBP mRNA was quantified by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) following inhibitory avoidance task in aged (26- to 27-month-old) rats that, according to memory performance, were ranked as good responders (GR) and bad responders (BR), in adult (3-month-old), late-adult (19-month-old), and aged (26-27-month-old) naive animals. We found that DNMBP mRNA levels were significantly higher in naive adults versus late adult and aged naive rats, in GR versus BR, and in pooled GR and BR versus aged-matched controls. Our data provide the first evidence that hippocampal DNMBP mRNA expression is reduced during physiological aging, and suggest that the capability to increase the expression of this mRNA may be a requirement for preserving long term memory formation during aging.

Physical training modulates structural and functional features of cell nuclei in type II myofibers of old mice.

Aging is associated with a progressive loss of muscle mass, strength, and function, a condition known as sarcopenia, which represents an important risk factor for physical disability in elderly. The mechanisms leading to sarcopenia are still largely unknown, and no specific therapy is presently available to counteract its onset or progress. Many studies have stressed the importance of physical exercise as an effective approach to prevent/limit the age-related muscle mass loss. This study investigated the effects of physical training on pre-mRNA pathways in quadriceps and gastrocnemius muscles of old mice by ultrastructural cytochemistry: Structural and in situ molecular features of myonuclei and satellite cell nuclei of type II fibers were compared in exercised versus sedentary old mice, using adult individuals as control. Our results demonstrated that in myonuclei of old mice physical exercise stimulates pre-mRNA transcription, splicing, and export to the cytoplasm, likely increasing muscle protein turnover. In satellite cells, the effect of physical exercise seems to be limited to the reactivation of some factors involved in the transcriptional and splicing apparatus without increasing RNA production, probably making these quiescent cells more responsive to activating stimuli.

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 A(2) (PLA(2)) 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.

Differences in gene expression in the hippocampus of aged rats are associated with better long-term memory performance in a passive avoidance test.

Microarray analysis was used to identify genes differentially expressed in the hippocampus of aged rats showing diverse long-term (3 and 6 h) spatial-associative memory performance in a single-trial inhibitory avoidance task. The transcription of 43 genes (including genes functionally linked to signal transduction, cell growth and differentiation, translation, energy metabolism, and nucleic acid processing) was significantly upregulated in good- versus bad-performing animals, whereas that of 18 genes (including genes functionally linked to transcription, cell growth and differentiation, apoptosis, and protein transport) was significantly downregulated in good- versus bad-performing animals. The differential expression of 14 of these genes was confirmed by real-time polymerase chain reaction.