Feeding melatonin enhances the phase shifting response to triazolam in both young and old golden hamsters

Aging alters many aspects of circadian rhythmicity, including responsivity to phase-shifting stimuli and the amplitude of the rhythm of melatonin secretion. As melatonin is both an output from and an input to the circadian clock, we hypothesized that the decreased melatonin levels exhibited by old hamsters may adversely impact the circadian system as a whole. We enhanced the diurnal rhythm of melatonin by feeding melatonin to young and old hamsters. Animals of both age groups on the melatonin diet showed larger phase shifts than control-fed animals in response to an injection with the benzodiazepine triazolam at a circadian time known to induce phase advances in the activity rhythm of young animals. Thus melatonin treatment can increase the sensitivity of the circadian timing system of young animals to a nonphotic stimulus, and the ability to increase this sensitivity persists into old age, indicating exogenous melatonin might be useful in reversing at least some age-related changes in circadian clock function.     

Effect of exogenous melatonin on viability, ingestion capacity, and free-radical scavenging in heterophils from young and old ringdoves (Streptopelia risoria)

The decrease of melatonin production with aging contributes to the decline in immune function as organisms age. Treatment with the exogenously administered indoleamine restores the reduced immunological functions. Therefore, we investigated the effect of melatonin on viability, phagocyte ingestion capacity, and free radical generation levels of heterophils from young and old ringdove (Streptopelia risoria) aged 3–4 and 11–13 years, respectively. Animals received a single oral dose of melatonin 1 h before lights off for three consecutive days. Experiments were performed at the acrophases and nadirs of melatonin. Melatonin treatment significantly increased serum melatonin levels at the acrophases, but not at the nadirs of the two age groups. In both young and old animals there was increased heterophil viability at acrophases with respect to nadirs, and also increased cell resistance to oxidative stress in the old animals after the melatonin treatment. At acrophases, the index, percentage and efficiency of phagocytosis all increased significantly, and superoxide anion levels decreased significantly with respect to the nadir values of vehicle and melatonin-treated animals, the effect being greater in young than in old ringdoves. At the nadirs, no change was observed in any parameter analyzed. In both young and old animals, phagocytosis and melatonin were positively correlated, while superoxide anion levels and melatonin were negatively correlated. In conclusion, exogenous melatonin enhanced heterophil viability in old animals as well as increasing phagocytosis and free-radical scavenging in both age groups during the nocturnal period, accompanied by an increase in the levels of the indoleamine.     

Age Related Changes from Youth to Adulthood in Rat Brain Cortex: Nitric Oxide Synthase and Mitochondrial Respiratory Function

Age related changes in brain cortex NO metabolism were investigated in mitochondria and cytosolic extracts from youth to adulthood. Decreases of 19%, 40% and 71% in NO production were observed in mitochondrial fractions from 3, 7, and 14 months old rats, respectively, as compared with 1-month-old rats. Decreased nNOS protein expression in 14 months old rats was also observed in mitochondria as compared with the nNOS protein expression in 1-month-old rats. Low levels of eNOS protein expression close to the detection limits and no iNOS protein expression were significantly detected in mitochondrial fraction for both groups of age. NO production in the cytosolic extracts also showed a marked decreasing tendency, showing higher levels than those observed in mitochondrial fractions for all groups of age. In the cytosolic extracts, however, the levels were stabilized in adult animals from 7 to 14 months. nNOS protein expression showed a similar age-pattern in cytosolic extracts for both groups of age, while the protein expression pattern for eNOS was higher expressed in adult rats (14 months) than in young animals. As well as in mitochondrial extracts iNOS protein expression was not significantly detected in cytosolic extracts at any age. RT-PCR assays indicated increased levels of nNOS mRNA in 1-month-old rats as compared with 14 months old rats, showing a similar pattern to that one observed for protein nNOS expression. A different aged pattern was observed for eNOS mRNA expression, being lower in 1-month-old rats as compared with 14 months old animals. iNOS mRNA was very low expressed in both groups of age, showing a residual iNOS mRNA that was not significantly detected. State 3 respiration rates were 78% and 85% higher when succinate and malate-glutamate were used as substrates, respectively, in 14 months rats as compared with 1-month-old rats. No changes were observed in state 4 respiration rates. These results could indicate 1 that nNOS and eNOS mRNA and protein expression can be age-dependent, and confirmed the nNOS origin for the mitochondrial NOS. During rat growth, the respiratory function seems to be modulated by NO produced by the different NOS enzymes: nNOS, eNOS and mtNOS present in the cytosol and in the mitochondria.     

The Bacteriostatic Protein Lipocalin 2 Is Induced in the Central Nervous System of Mice with West Nile Virus Encephalitis

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.     

Comparative Study of the Activity/Rest Rhythms in Young and Old Ringdove (Streptopelia Risoria): Correlation with Serum Levels of Melatonin and Serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2–3 and 10–12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12∶12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09∶00 to 18∶00 h) and 1 h (from 21∶00 to 06∶00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02∶00 and 14∶00 h in the young and 01∶00 and 13∶00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one‐third that of the young animals. The acrophase and nadir were at 15∶00 and 03∶00 h in the young and 16∶00 and 04∶00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age‐associated sleep.     

Comparative study of the activity/rest rhythms in young and old ringdove (Streptopelia risoria): correlation with serum levels of melatonin and serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2-3 and 10-12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12:12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09:00 to 18:00 h) and 1 h (from 21:00 to 06:00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02:00 and 14:00 h in the young and 01:00 and 13:00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one-third that of the young animals. The acrophase and nadir were at 15:00 and 03:00 h in the young and 16:00 and 04:00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age-associated sleep.     

Influence of age on brain edema formation, secondary brain damage and inflammatory response after brain trauma in mice

After traumatic brain injury (TBI) elderly patients suffer from higher mortality rate and worse functional outcome compared to young patients. However, experimental TBI research is primarily performed in young animals. Aim of the present study was to clarify whether age affects functional outcome, neuroinflammation and secondary brain damage after brain trauma in mice. Young (2 months) and old (21 months) male C57Bl6N mice were anesthetized and subjected to a controlled cortical impact injury (CCI) on the right parietal cortex. Animals of both ages were randomly assigned to 15 min, 24 h, and 72 h survival. At the end of the observation periods, contusion volume, brain water content, neurologic function, cerebral and systemic inflammation (CD3+ T cell migration, inflammatory cytokine expression in brain and lung, blood differential cell count) were determined. Old animals showed worse neurological function 72 h after CCI and a high mortality rate (19.2%) compared to young (0%). This did not correlate with histopathological damage, as contusion volumes were equal in both age groups. Although a more pronounced brain edema formation was detected in old mice 24 hours after TBI, lack of correlation between brain water content and neurological deficit indicated that brain edema formation is not solely responsible for age-dependent differences in neurological outcome. Brains of old naïve mice were about 8% smaller compared to young naïve brains, suggesting age-related brain atrophy with possible decline in plasticity. Onset of cerebral inflammation started earlier and primarily ipsilateral to damage in old mice, whereas in young mice inflammation was delayed and present in both hemispheres with a characteristic T cell migration pattern. Pulmonary interleukin 1β expression was up-regulated after cerebral injury only in young, not aged mice. The results therefore indicate that old animals are prone to functional deficits and strong ipsilateral cerebral inflammation without major differences in morphological brain damage compared to young.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Developmental pattern of tachykinins during aging in several organs: effect of exogenous melatonin

Mammalian neurokinin A (NKA) and substance P (SP) are neuropeptides widely distributed in the body; they are potential regulators of the basal blood flow and therefore of the function of many organs and tissues. In the present investigation, we studied the age-dependent changes in NKA and SP in ovary, liver, pancreas and spleen as well as the role of exogenous melatonin on these changes. Female rats of 5, 15 or 25 months of age were studied. In the ovary, NKA concentrations did not change during aging. SP concentrations in the control group were significantly higher (P<0.01) in old rats than in the other two age groups studied. Melatonin treatment resulted in reduced concentrations as compared with those of the control old rats. In the pancreas, NKA and SP concentrations increased during aging, the young rats showing significantly lower values (P<0.01) than middle-aged and old rats for NKA and significantly lower (P<0.01) than the old rats for SP. After melatonin treatment the differences in NKA concentrations disappeared and SP decreased in middle-aged as compared with those in old rats. In the liver, NKA and SP concentrations in the control and melatonin-treated groups did not differ significantly for the three age groups studied. Splenic NKA in control and melatonin-treated groups increased from young to middle-age up to old ages. SP concentrations showed similar values at all ages except in melatonin-treated old rats; in these animals there were significantly higher concentrations than in young melatonin-treated rats. The effect of melatonin was mainly observed on the ovary and pancreas in old rats, with a reduction in the concentrations as compared with those observed in the young groups.     

Protective effect of xanthohumol against age-related brain damage

It has been recently shown that xanthohumol, a flavonoid present in hops, possesses antioxidant, anti-inflammatory and chemopreventive properties. However, its role in the aging brain has not been addressed so far. Therefore, this study aimed to investigate the possible neuroprotective activity of xanthohumol against age-related inflammatory and apoptotic brain damage in male senescence-accelerated prone mice (SAMP8). Animals were divided into 4 groups: Untreated young mice, untreated old mice and old mice treated either with 1 mg kg⁻¹ day⁻¹ or 5 mg kg⁻¹ day⁻¹ xanthohumol. Young and old senescence accelerated resistant mice (SAMR1) were used as controls. After 30 days of treatment, animals were sacrificed and their brains were collected and immediately frozen in liquid nitrogen. mRNA (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, XIAP, NAIP and Bcl-2) and protein (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, BDNF, synaptophysin and synapsin) expressions were measured by RT-PCR and Western blotting, respectively. Significant increased levels of pro-inflammatory (TNF-α, IL-1β) and pro-apoptotic (AIF, BAD, BAX) markers were observed in both SAMP8 and SAMR1 old mice compared to young animals (P<.05) and also in SAMP8 untreated old mice compared to SAMR1 (P<.05). These alterations were significantly less evident in animals treated with both doses of xanthohumol (P<.05). Also, a reduced expression of synaptic markers was observed in old mice compared to young ones (P<.05) but it significantly recovered with 5 mg kg⁻¹ day⁻¹ xanthohumol treatment (P<.05). In conclusion, xanthohumol treatment modulated the inflammation and apoptosis of aged brains, exerting a protective effect on damage induced by aging.     

Melatonin, lipid peroxidation, and age in heterophils from the ring dove (Streptopelia risoria)

Numerous recent studies have shown the ability of physiological as well as all pharmacological concentrations of melatonin to prevent oxidative stress. We have found that incubating avian heterophils from young birds with a pharmacological concentration of 100 μM (23 × 10⁶ pg/ml) melatonin reduced superoxide anion levels by modulating the activity of superoxide dismutase while also enhancing phagocytosis. There was also a decline in lipid peroxidation levels with both physiological and pharmacological concentrations of this indolamine.

In the present work, we evaluated malonaldehyde (MDA) levels as an indicator of lipid peroxidation (both basal and antigen-induced) in young and old animals (ring doves) at different times of day (16:00 and 00:00) and with two incubation times (15 and 60 min). The lipid peroxidation was also measured in heterophils from old animals, incubated with the physiological concentrations of melatonin measured in young animals (50 and 300 pg/ml, diurnal and nocturnal, respectively). The results, expressed as nmol MDA/mg protein, show that MDA levels were higher in heterophils of old animals than in the young birds in all the experimental groups studied at both 16:00 and 00:00 (00:00 is the time at which the lowest peroxidation levels were obtained). Incubation with melatonin was found to reduce MDA levels, with the maximum reduction being after the 60 min incubation time and the nocturnal melatonin concentration. At both concentrations (diurnal and nocturnal), melatonin also counteracted the enhancement of MDA levels caused by latex beads, with the effect being greater at the longer incubation time. In conclusion, the results are further evidence of the antioxidant effect of melatonin even at physiological concentrations, and suggest its utility as a therapeutic agent in some pathological processes associated with age.     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Ageing,opioid analgesia and the pineal gland

The effects of ageing on day-night rhythms of analgesia was examined with young (1–2 months), mature (8–12 months) and old (20–30 months) mice. Significant age-related declines were observed both in the absolute levels and diel rhythms of morphine analgesia, with the most pronounced changes occuring at night. Administration of the pineal hormone, melatonin, augmented day-time levels of analgesia in all age classes and reversed the age-related decline in nocturnal morphine analgesia in old mice. Inhibition of pineal function in young mice by either exposure to light pulses or treatment with benserazide mimicked the effects of ageing on nocturnal morphine analgesia. These findings suggest that the pineal gland and melatonin are involved in modulating diel rhythms of analgesia and have an influential role on age-related changes in opioid responses.     

Lack of effects of 1439 MHz electromagnetic near field exposure on the blood-brain barrier in immature and young rats

Possible effects of 1439 MHz electromagnetic near field (EMF) exposure on the blood-brain barrier (BBB) were investigated using immature (4 weeks old) and young (10 weeks old) rats, equivalent in age to the time when the BBB development is completed and the young adult, respectively. Alteration of BBB related genes, such as those encoding p-glycoprotein, aquaporin-4, and claudin-5, was assessed at the protein and mRNA levels in the brain after local exposure of the head to EMF at 0, 2, and 6 W/kg specific energy absorption rates (SARs) for 90 min/day for 1 or 2 weeks. Although expression of the 3 genes was clearly decreased after administration of 1,3-dinitrobenzene (DNB) as a positive control, when compared with the control values, there were no pathologically relevant differences with the EMF at any exposure levels at either age. Vascular permeability, monitored with reference to transfer of FITC-dextran, FD20, was not affected by EMF exposure. Thus, these findings suggest that local exposure of the head to 1439 MHz EMF exerts no adverse effects on the BBB in immature and young rats.