Nutritional antioxidants as antidegenerative agents.

In this study, primary cultures of cerebellar granule neurons were prepared from eight-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. All experiments were performed with fully differentiated neurons (eight days). To induce apoptosis, culture medium was replaced with a serum-free medium (containing 5 mM KCl) eight days after plating. In another series of experiments, apoptosis was induced by application of glutamate (50 microM) to the cell cultures. Apoptosis was measured by flow cytometry, the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling) method, and by the classical method of DNA fragmentation. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by both low K(+) concentrations and glutamate, a series of natural antioxidants and a red wine lyophilized extract (which is rich in antioxidant compounds) were tested in our experimental model. It was found that ascorbic acid (30 microM) and a red wine lyophilized extract (5 microgram/ml) were capable of blocking the apoptotic process. Addition of the following natural antioxidants did not have any protective effect on apoptosis induced by low K(+) concentrations: trans- and cis-resveratrol (5-200 microM), alpha-tocopherol (100-200 microM), reduced glutathione (100-400 microM), 3-hydroxytirosol (25-100 microM), epicatechin (25-100 microM), or quercetin (25-50 miroM). It is concluded that only a limited number of natural antioxidants are provided with antiapoptotic activity in cultured cerebellar granule neurons. This effect is probably exerted by reducing ROS formation, and by blocking caspase-3 activity.

Subunit and region-specific decreases in nicotinic acetylcholine receptor mRNA in the aged rat brain.

We have investigated possible changes in the mRNA levels for several alpha and beta subunits of the nicotinic acetylcholine receptor (nAChR) and the level of binding for nicotinic ligands in 7- to 32-month-old rats. Alpha4 and beta2, and to a lesser extent alpha6 and beta3, mRNA levels showed decreases between 20 and 30% at 29 months of age which in some areas reached 50% at 32 months of age. Alpha7 showed a small increase from 7 to 14 months and then a progressive decrease from 14 to 32 months down to the 7-month levels. 3H-epibatidine binding did not significantly change from 7 to 32 months of age in rat tel- and diencephalon. Binding in the substantia nigra was exceptional in that it showed a significant decrease starting from 23 months of age. 125I-alpha-bungarotoxin binding showed a pattern of change which roughly paralleled that of alpha7 mRNA. These findings show that an alteration in some steps of nAChR biosynthesis takes place during aging, which may be related to functional changes in nicotinic transmission.

The effect of caloric restriction on the aortic tissue of aging rats.

Connective tissue shows peculiar and complex age-related modifications, which can be, at least in part, responsible for altered functions and increased susceptibility to diseases. Food restriction has long been known to prolong life in rodents, having antiaging effects on a variety of physiologic and pathologic processes. Therefore, the aorta has been investigated in rats fed normal or hypocaloric diet, from weaning to senescence. Compared with controls, caloric-restricted animals showed less pronounced age-dependent alterations such as elastic fiber degradation, collagen accumulation and cellular modifications. Immunocytochemical analyses revealed that elastic fibers were positively labelled for biglycan, decorin, ApoB100 (LDL), ApoA1 (HDL) and elastase and that the intensity of the reactions was time- and diet-dependent. With age, the major changes affecting aortic elastic fibers were increased positivity for decorin, LDL and elastase. Compared with age-matched normal fed rats, caloric restricted animals revealed lower content of LDL, decorin and elastase and higher positivity for HDL. These data suggest that a caloric restricted diet might influence the aging process of the arterial wall in rats, delaying the appearance of age-related degenerative features, such as structural alterations of cells and matrix and modified interactions of elastin with cells and with other extracellular matrix molecules.

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.

A reduced calorie-high fiber diet retards age-associated decreases in muscarinic receptor sensitivity.

The effects of a reduced calorie-high fiber diet (RCHF) were examined on three cholinergic signal transduction (ST) parameters: (a) oxotremorine enhancement of K(+)-evoked dopamine release and (b) carbachol-stimulated low KM GTPase activity [an indicator of muscarinic receptor (mAChR)-G protein coupling/uncoupling], and (c) [3H]Quinuclidinyl benzilate (QNB) autoradiography. Comparisons were made among: young control (6 months), old normal control, old reduced calorie high fiber [both 24 months)]. The results indicated that old reduced calorie high fiber rats (1900 kcal/kg/day, 2.4%, lipids 2.4%, fiber 28%, carbohydrates 40.7%) as compared to the old normal control rats (3000 kcal/kg/day, 4.8% lipids, 4.2% fiber, carbohydrates 61.5%) showed a retardation of age-related deficits in dopamine release (a above) and GTPase activity (b above). These parameters were 25% higher in the old reduced calorie high fiber rats as compared to old normal controls and did not differ from young controls, even though there was no increase in mAChR concentration in the restricted group. Thus, these results indicate that a reduced calorie high fiber diet as utilized in these experiments was effective in retarding the age-related decrements in two of three signal transduction parameters. They are discussed in terms of the induction of membrane changes (e.g., fluidity) or related decreases in oxidative stress by the restricted diet that may be involved in these signal transduction effects.

Aging prolongs the stress-induced release of noradrenaline in rat hypothalamus.

A stress-induced increase in noradrenaline (NA) release was measured by intracerebral microdialysis in the hypothalamic paraventricular nucleus of freely moving Wistar-Kyoto rats at three different ages (6, 18 and 24 months). NA levels in 20-min dialysate samples were measured by high-performance liquid chromatography with electrochemical detection. Microdialysis sampling was done at the baseline during a 20-min immobilization stress and for the next 100 min. Basal NA release was not significantly different in the three age groups. The immobilization stress increased NA levels (247, 197 and 234% of the baseline for the 6-, 18- and 24-month animals, respectively) which was not significantly different in the three groups. In the two younger groups NA returned to the baseline in the first sample after the end of the stress (t = 40 min) whereas in the 24-month group it remained significantly higher for longer (until t = 60 min). Stress-induced release of hypothalamic NA thus appears to be prolonged in old rats.

Effect of oxiracetam on scopolamine-induced amnesia in the rat in a spatial learning task.

The effects of the nootropic agent 4-hydroxy-2-oxopyrrolidinoacetamide (oxiracetam) on memory and performance impairments induced by scopolamine were evaluated in the Morris water maze task. No effect was seen on the performance of rats when treated with oxiracetam (30 mg/kg, IP) alone. Task performance of scopolamine (0.2 mg/kg, SC)-treated rats was impaired as compared to that of control animals. The behavioral deficits expressed in the task by scopolamine treatment were attenuated by the same dose of oxiracetam.

Correlation between electroencephalogram isoelectric time and hippocampal norepinephrine levels, measured by microdialysis, during ischemia in rats.

It is suggested that norepinephrine (NE) plays a role during transient forebrain ischemia. NE may have a protective action against neuronal cell death in the hippocampus, or it may be one of the causes of injurious ischemic effects. We used the microdialysis technique to study extracellular NE levels in the rat hippocampus before, during, and after 30 min of transient incomplete forebrain ischemia (induced by four-vessel occlusion) to describe the time course of NE in this condition. There was a maximal increase (fivefold) in extracellular NE after 10 min of reflow only when the electroencephalogram was isoelectric. NE levels returned to baseline 40 min after release of the carotid clamps and remained constant for the next 80 min. Thus there appears to be a transient NE overflow in the hippocampus during ischemia, closely related to the complete loss of brain electrical activity.

Deterioration of spatial and nonspatial reference and working memory in aged rats: protective effect of life-long calorie restriction.

Two different aspects of learning (spatial and nonspatial) and two different types of memory (reference and working) were simultaneously measured in populations of 3- (young), 11- (adult), and 25-month-old (aged) rats fed ad libitum either a standard (ST) or a hypocaloric (HY) diet. All groups, regardless of age or diet, showed ability in learning all four versions of the task. However, old ST rats were significantly slower and less efficient at learning than the young and adult ST rats. In contrast, senescent HY rats' cognitive abilities did not differ from those of their young and adult counterparts. The decline in reference and working memory in the aged ST rats was more pronounced in the spatial than the nonspatial version of the task. This study confirms and extends to more specific aspects of memory our earlier finding that age-related cognitive deterioration in rats was antagonized by life-long calorie restriction.

Age-related ability of rats kept on a life-long hypocaloric diet in a spatial memory test. Longitudinal observations.

Cognitive behavior was assessed in a test specific for spatial memory, according to a longitudinal experimental model, in populations of 12-, 24- and 30-month-old rats which were fed ad lib either a standard (ST) or a hypocaloric (HY) diet, that had been shown to reduce age-related pathologies and to increase survival. Already at 12 months of age, some cognitive deficits were observed in ST but not in HY rats. When retested 12 and 18 months later, the animals performed better at the beginning of the test than when tested for the first time, indicating that some aspects of previous experience lead to a preservation of spatial memory. Deficits which had been previously observed in 2-year-old groups in a cross-sectional experimental model were not evident this time. Some of the differences between the two diet groups observed 12 months before disappeared. When testing was repeated for the third time at 30 months of age, ST fed rats presented very marked deficits in learning and in memory, which were not seen in the HY group. It thus appears that a dietary regimen in which part of the calorie-rich components, such as lipids and carbohydrates, are replaced with vegetable fibers, retards some of the age-related deteriorations of brain functions.

Effect of lifelong hypocaloric diet on discrete memory of the senescent rat.

The memory retention abilities of aged rats fed different diets were assessed in two different avoidance tasks. The standard passive avoidance procedure revealed an age-related memory impairment in old rats fed a standard diet (ST), whereas old rats fed a hypocaloric diet (HY) behaved similarly to young animals. To clarify whether this deficit could be attributed only to cognitive decay and not other factors, such as the tendency of old rats to prefer darkness to light more than young and adult animals, a multiple passive avoidance task was performed. This test offers rats the possibility to escape to a dark chamber in which they have never been shocked, and thus provides a means of checking factors other than memory retention abilities. All the old rats showed a more marked preference to escape to darkness compared to young and adult animals. However, senescent animals fed a ST diet had poor memory retention abilities compared to aged animals fed the HY diet, and young and adult rats. The results of this test confirmed the findings of the standard passive avoidance task.

Previous experience facilitates preservation of spatial memory in the senescent rat.

In an attempt to evaluate whether previous training antagonizes age-related memory deficits, aged rats with previous training experience were compared with aged and young naive rats in the Morris water maze test. The findings of our study indicate that experience seems to facilitate the preservation of the spatial reference memory for 12 months in the aged rat, whereas senescent naive animals showed the usual age-related memory deficits. However, experience seems task-specific since the same aged rats fail to acquire a new different task.

Effect of Org2766, an ACTH(4-9) analogue, on recovery after bilateral transection of the fimbria fornix in the rat.

Facilitation of recovery after peripheral or central damage of the nervous system induced by neuropeptides was recently extensively reported. In a previous study we reported that Org2766, an ACTH(4-9) analogue, enhances recovery as assessed in a spatial orientation task after unilateral transection of the fimbria fornix in the rat. It was suggested that cross-lateral compensation by the intact fimbria fornix hippocampal system could account for the peptide-induced recovery. Therefore, the facilitatory effect of this neuropeptide was investigated in the present study after bilateral transection of the fimbria fornix. The present results indicate that Org2766 also attenuated the behavioral deficit of bilaterally transected animals in a spatial learning task, but does not affect behaviour of the lesioned animals in a passive avoidance test.

Aging and food restriction: effect on lipids of cerebral cortex.

In experimental animals dietary restriction reduces the body weight increase due to aging, increases longevity and delays the onset of age-related physiological deterioration, including age-related changes in serum lipids. Little is known about the influence of food restriction on brain lipids, whose concentration and composition have been shown to change with age. We studied whether some biochemical and biophysical parameters of rat brain membranes, known to be modified with age, were affected by a diet low in calories, in which 50% of lipids and 35% of carbohydrates have been replaced by fibers. The diet was started at weaning and maintained throughout the animal's entire life span. Animals fed the low calorie diet survived longer and gained less body weight than standard diet fed rats. Age-related increases in microviscosity, cholesterol/phospholipid and sphingomyelin/phosphatidylcholine ratios were reduced or restored to the levels of young animals in cortex membranes of 32 old rats fed the low calorie diet, while the age-related increase in mono- to polyunsaturated fatty acid ratios in phospholipids was further raised. In conclusion we have shown that a diet low in calories and high in fibers affects lipid composition in the rat brain, in a direction opposite to that normally believed to reduce age-related deterioration of brain functions.

The ACTH/MSH (4-9) analog Org2766 improves retrieval of information after a fimbria fornix transection.

The fimbria fornix of male Wistar rats was transected unilaterally after they had been successfully trained in the Morris maze and the passive avoidance task. Sham-operated and lesioned animals were treated either with Org2766 or saline for two weeks. Subsequently, the performance of all groups was tested again starting two days after the last treatment. The lesioned animals showed a deficit in performance in both tasks, indicating interference of the lesion with retrieval of information. Org2766 improved the poor performance of the lesioned animals in the Morris maze, but not in the passive avoidance task.

Org2766 improves performance of rats with unilateral lesions in the fimbria fornix in a spatial learning task.

ACTH and related peptides are known for their effects on behavior and for their efficacy on peripheral nerve regeneration. More recently, evidence demonstrating neurotrophic effects of Org2766 on recovery after damage of the central nervous system has become available. Aging studies indicated that particularly the hippocampus appears susceptible to peptide treatment. Therefore, in this study the neurotrophic influence of Org2766 on recovery of hippocampal functioning was studied after transection of the fimbria fornix connection. Analogous to results obtained with recovery of sensorimotor function following peripheral nerve damage a chronic treatment with Org2766 facilitates recovery of the impaired function, i.e. spatial learning in a Morris maze. However, the lesion-induced impairment of avoidance behavior was not influenced by the peptide treatment.