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The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35–75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

Ageing of the human immune system, or immunosenescence, is characterised by distinct changes in the proportion of the various cell types, e.g., increase of the CD14+ monocytic cells, decrease of CD19+ B lymphocytes, and changes in T cell subpopulations, namely increase of CD4+ and CD8+ cells which have lost the costimulatory CD28 antigen. Currently, it is believed that the lifelong antigenic burden may be one of the inducers of immunosenescence. Thus far, only one exogenous stimulus, cytomegalovirus infection, has shown to be a major factor in this respect. To find other possible candidates, we evaluated the role of the evolutionary youngest group of human endogenous retroviruses, namely HERV-K(HML-2), on immunosenescence. HERVs exist in the genome as proviruses, but their activation has been detected in several immunopathologic conditions. The expression of HERV-K(HML-2) env was observed to be lower in the peripheral blood mononuclear cells of nonagenarians (n=61) than in those of young controls (n=37). These mRNA levels did not correlate with the age-associated differences in the proportions of CD14+, CD4+CD28- and CD8+CD28- cells, but in the case of CD19+ B cells a strong positive correlation was observed in the nonagenarians. Thus, these data suggest that HERVs do not function as antigenic drivers of immunosenescence. On the contrary, expression of HERV-K(HML-2) env is associated with more youthful levels of B cells.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

Epigenetic mechanisms such as DNA methylation (DNAm) have a central role in the regulation of gene expression and thereby in cellular differentiation and tissue homeostasis. It has recently been shown that aging is associated with profound changes in DNAm. Several of these methylation changes take place in a clock-like fashion, i.e. correlating with the calendar age of an individual. Thus, the epigenetic clock based on these kind of DNAm changes could provide a new biomarker for human aging process, i.e. being able to separate the calendar and biological age. Information about the correlation of the time indicated by this clock to the various aspects of immunosenescence is still missing. As chronic cytomegalovirus (CMV) infection is probably one of the major driving forces of immunosenescence, we now have analyzed the correlation of CMV seropositivity with the epigenetic age in the Vitality 90+cohort 1920 (122 nonagenarians and 21 young controls, CMV seropositivity rates 95% and 57%, respectively). The data showed that CMV seropositivity was associated with a higher epigenetic age in both of these age groups (median 26.5 vs. 24.0 (p < 0.02,Mann–Whitney U-test) in the young controls and 76.0 vs. 70.0 (p < 0.01) in the nonagenarians). Thus, these data provide a new aspect to the CMV associated pathological processes.

Background Ageing is characteristically accompanied by changes in vascular endothelial markers and growth factor as well as increased cellular death. We analysed the associations of the plasma levels of vascular cell adhesion molecule-1 (VCAM-1), hepatocyte growth factor (HGF) and soluble Fas (sFAS), and their combinations, with 4-year mortality to identify new biomarkers. Methods A total of 238 individuals, both community-dwelling and institutionalised, aged 89‒91 years and participating in the Vitality 90 + study were included. Biomarkers of endothelial function (VCAM-1), growth factor (HGF) and a marker of apoptosis (sFAS) were determined from plasma using Luminex® technology. This newly-determined data was combined with earlier data, e.g., 4-year mortality and medical history. Results Subjects who died during the follow-up had higher baseline plasma levels of VCAM-1, sFas, and HGF. When other known risk factors were adjusted for, subjects in the highest concentration tertile for VCAM-1 (HR 1.85; 95% CI, 1.12–3.05) and HGF (HR 2.22; 95% CI, 1.33–3.71) had higher mortality compared to those in the lowest tertile. In the adjusted analyses, when compared to subjects with none of the biomarkers in the highest concentration tertile, mortality was also higher when sFas and VCAM-1 were simultaneously (HR 2.03; 95% CI, 1.13–3.64) or all three were simultaneously (HR 3.63; 95% CI, 1.65–7.97) in the highest concentration tertile. Conclusions Our results suggest that increased concentrations of these biomarkers, separately and in combination, associate with mortality among the aged and are prognostic markers of death.

The purpose of the present study was to examine the combined effects of aging and lifelong ethanol exposure on the levels of monoamine neurotransmitters in different regions of the brain. This work is part of a project addressing interactions of aging and lifelong ethanol consumption in alcohol-preferring AA (Alko Alcohol) line of rats, selected for high voluntary consumption of ethanol. Intake of ethanol on the level of 4.5–5 g/kg/day for about 20 months induced only limited changes in the neurotransmitter levels; the concentration of noradrenaline was significantly reduced in the frontal cortex. There was also a trend towards lower levels of dopamine and 5-hydroxytryptamine (5-HT) in the frontal cortex, and towards a lower noradrenaline level in the dorsal cortex. Aging was associated with a decreased concentration of dopamine in the dorsal cortex and with a declining trend in the striatum. The levels of 5-HT in the limbic forebrain were higher in the aged than in the young animals, and in the striatum, there was a trend towards higher levels in older animals. The data suggest that a continuous intake of moderate amounts of ethanol does not enhance the age-related alterations in brain monoamine neurotransmission, while the decline in the brain level of dopamine associated with aging may be a factor contributing to age-related neurological disorders.

Increased proinflammatory status is associated with both increased adiposity and higher mortality risk. Thus, it is paradoxical that mild obesity does not predict increased mortality in older adults. We investigated the association of inflammatory markers with body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) in nonagenarians, and the combined effects of BMI, WC, WHR, and inflammatory status on mortality.This study was based on a prospective population-based study, Vitality 90+, carried out in Tampere, Finland. Altogether, 157 women and 53 men aged 90 years were subjected to anthropometric measurements, blood samples, and a 4-year mortality follow-up. Inflammatory status was based on sex-specific median levels of interleukin-1 receptor antagonist (IL-1RA), interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α).In the unadjusted linear regression analyses, IL-1RA, CRP, and TNF-α were positively associated with BMI and WC in women, whereas in men IL-1RA was positively associated with BMI and IL-6 positively with WC. In the models adjusted for diseases, functional status, and smoking, IL-1RA and CRP were positively associated with BMI and WC in women. Low WC and WHR combined with low inflammation protected from mortality in women and high BMI and WC regardless of inflammation protected from mortality in men in the adjusted Cox regression analysis.In the oldest old, the effect of adiposity in combination with inflammatory status on mortality differs between men and women. More research is needed to disentangle the role of adiposity among the oldest old.

Aging is characteristically accompanied by a low-grade inflammatory state and increased cellular death. In this study, we analyzed the associations between the plasma cell-free DNA (cf-DNA) level and immunoinflammatory factors and 4-year mortality. The distribution of qualitative cf-DNA patterns in relation to the total cf-DNA concentration and the antinuclear antibody (ANA) serostatus was also evaluated. The study population consisted of n=258 nonagenarians who were participants in the Vitality 90+ Study. Cf-DNA levels were positively correlated with the C-reactive protein (CRP) level, the interleukin-1 receptor antagonist (IL-1ra) level, the serum amyloid A (SAA) level and the indoleamine 2,3-dioxygenase enzyme activity (IDO), weight and chair-stand test time and inversely correlated with the HDL cholesterol level. The total cf-DNA concentration also remained as an independent predictor of 4-year all-cause mortality. A predominance of low-molecular-weight cf-DNA was observed in ANA-seropositive subjects but this cf-DNA pattern was not associated with mortality. The cf-DNA concentration could thus represent a novel biomarker for systemic inflammation and mortality in the elderly.

OBJECTIVES: To establish whether the relationship between interleukin-6 (IL-6) and plasma lipid and C-reactive protein (CRP) concentrations is different in Finnish nonagenarians than in middle-aged subjects with lower inflammatory status. DESIGN: Cross-sectional. SETTING: Observational cohort study concentrating on the oldest old. PARTICIPANTS: Nonagenarians (n = 291, mean age ± standard deviation 90 ± 1; 68 men, 223 women) who lived in the Tampere municipality in southern Finland and a middle-aged control population from the same area (n = 227, aged 44 ± 8). MEASUREMENTS: Plasma high sensitive CRP and lipid concentrations were analyzed using an automatic analyzer and IL-6 levels using enzyme-linked immunosorbent assay. RESULTS: Plasma concentrations of IL-6 (4.39 ± 5.25 vs 1.88 ± 1.98 pg/mL) and CRP (3.54 ± 4.98 vs 1.53 ± 1.91 mg/L) were significantly higher in nonagenarians than in middle-aged subjects (P

Data on how body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) are associated with body fat in the oldest-old people are scarce. The purpose of this study was to examine if BMI, WC or WHR are associated with leptin, a biological surrogate measure of body fat in 90-year-old people. The data comes from the Vitality 90+ Study, a prospective population-based study of people living in Tampere, Finland. BMI, WC, WHR and plasma concentration of leptin were available for 160 women and 54 men aged 90 years. BMI and WC had a strong significant positive association with leptin both in women and in men, but WHR was associated with leptin only in men. In conclusion, based on the circulating level of leptin, BMI and WC, and WHR in men, reflect body fat in 90-year-old people, but WHR seems to be a poor indicator of body fat in 90-year-old women.

Polymorphisms affecting the expression of matrix metalloproteinases (MMPs), i.e. proteolytic enzymes that degrade intercellular material, have been found at position -1607 (1G/2G) in MMP1 and at -1171 (5A/6A) in MMP3. Interestingly, elevated levels of MMP1 and MMP3 have been observed in the brains of Alzheimer's disease (AD) patients and those of tissue inhibitors of MMPs in the cerebrospinal fluid of AD and Parkinson's disease (PD) patients, suggesting a role for MMPs in these disorders. The aim was to investigate a possible association between the afore-mentioned MMP1 and MMP3 polymorphisms and the risk of developing AD or PD. The polymorphisms were genotyped in 97 AD, 52 PD and 101 control patients. We found an interaction between MMP3*5A and APOE 4 alleles (P < 0.0001) which increases the risk of AD (OR: 23.7, 95% CI: 5.8-144.9, P < 0.0001) compared to those who possess neither MMP3*5A nor APOE 4. In conclusion, our finding suggests that the MMP3 gene, especially together with APOE 4, may contribute to the development of AD.

The effects of aging and lifelong ethanol consumption on astrocytic morphology and glial fibrillary acidic protein-immunoreactivity (GFAP-IR) in the cerebellar vermis obtained from ethanol-preferring Alko, Alcohol (AA) rats were analyzed by using computer-assisted image analysis. The ethanol-consuming animals (both male and female) were given ethanol (10%–12%, vol./vol.) as the only available fluid for 21 months (3–24 months), whereas the young (3 months) and the old (24 months) controls received water. In the male rats, but not in the female rats, an age-related decrease in GFAP-IR was found in folia II, VII, and X of the molecular layer, and in turn, an age-related increase was found in folium X of the granular layer, indicating opposite changes in GFAP-IR for male rats due to aging in adjacent brain regions. In the female rats, 21 months of daily average ethanol consumption of 6.6 g/kg resulted in decreased GFAP-IR in folium VII of the molecular layer, and the decrease in cerebellar GFAP-IR correlated with the average daily ethanol intake ( r =−.886, P =.019) when folia II, IV, VII, and X were analyzed together. No effect of ethanol on GFAP-IR was detected in the granular layer or in the central white matter of the female rats. There was no change in GFAP-IR in any of the three cerebellar layers of the male rats with average daily ethanol consumption of 3.2 g/kg. These results indicate that the Bergmann glial fibers are the GFAP-expressing structures of the cerebellum most sensitive to moderate-to-heavy chronic ethanol exposure and that this effect is dose dependent.

Background: Binge ethanol exposure is known to induce degeneration of central nervous system (CNS) neurons. Sympathetic hyperactivity has been related to ethanol withdrawal symptoms, but the effects of repeated withdrawals on peripheral sympathetic neurons have not been studied previously. Methods: The effects of continuous versus intermittent ethanol consumption on sympathetic neurons of the superior cervical ganglion (SCG) were studied in male Wistar rats. Two-month-old rats were divided into three groups: one group with ethanol (10% v/v) as the drinking fluid throughout the 5½-month experiment (continuous, n=9), one group drinking ethanol on 4 days/week and water on 3 days/week (intermittent, n=9), and a control group (n=9) with water as the only available fluid. All groups had food ad libitum. SCG volume, neuron density, and total number of neurons were measured by using unbiased morphometric methods. Results: As the mean daily ethanol consumption did not differ between the two ethanol-exposed groups (continuous 5.7 g/kg/day versus intermittent 5.8 g/kg/day), the total dose of ethanol consumed was 42% smaller in the intermittent group. The total number of SCG neurons decreased by 28%, and neuron density by 23%, in the intermittent group compared with the control group, whereas no significant neuron loss was observed in the continuous group. The volume of the SCG was similar in all study groups. The results suggest that repeated ethanol withdrawals, rather than ethanol exposure per se, are deleterious to sympathetic neurons. Conclusions: Ethanol-induced degeneration of neurons is not only related to the amount of ethanol consumed, but also to the patterns of drinking.

In the present study, the neuroprotective effects of dexmedetomidine on rat locus coeruleus were studied during a 5-week intermittent ethanol exposure. Male Wistar rats (3 to 4 months old) were given ethanol or isocaloric sucrose by intragastric intubations three times a day for 4 days, which was followed by a 3-day withdrawal period. This 7-day cycle of ethanol exposure and withdrawal was repeated five times. Dexmedetomidine (at a dose decreasing from 30 microg/kg to 10 microg/kg, s.c.) was given to the treatment group during the withdrawal phase. The results showed that, during the 5-week experiment, dexmedetomidine significantly relieved the ethanol withdrawal syndrome, measured as the sum of the three most specific symptoms (rigidity, tremor, and irritability). The total neuron number of locus coeruleus (LC) decreased in the ethanol-treated group by 24%, compared with the nontreated control group and by 11%, compared with the sucrose-treated control group. Interestingly, the LC neuron numbers were found to decrease in the sucrose-intubated rats as well, compared with the nontreated control group. Dexmedetomidine was found to relieve ethanol-induced neuronal loss in the LC. Dexmedetomidine might be a new interesting alternative in the treatment of ethanol withdrawal syndrome, particularly due to its possible neuroprotective effects in the central nervous system.

The aim of this work was to evaluate the effects of long-term ethanol consumption on arterial responses in vitro in young and aged rats. Therefore, Wistar rats (ages 3 and 29 mo, respectively) were allocated to six groups: control-young, sucrose-young, ethanol-young, control-aged, sucrose-aged, and ethanol-aged. The ethanol-fed groups were given 25% ethanol by intragastric gavage three times a day 4 days a week. Responses of mesenteric arterial rings were examined in standard organ chambers after 5 treatment weeks. In norepinephrine-precontracted arterial rings, endothelium-dependent relaxations to acetylcholine, as well as endothelium-independent relaxations to isoproterenol, were attenuated in aged rats when compared with young controls. Relaxation responses to isoproterenol, but not to acetylcholine and nitroprusside, were clearly improved by ethanol treatment in both young and aged rats. The cyclooxygenase inhibitor diclofenac, which reduces the synthesis of dilating and constricting prostanoids, enhanced the relaxation to acetylcholine in all three aged rat groups but was without significant effect in the young rats. In the presence of the nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester the relaxation to acetylcholine in control and sucrose-fed aged rats was markedly reduced compared with control rats, whereas in the young controls and in both young and aged ethanol-exposed groups, distinct relaxations to higher concentrations of acetylcholine were still present. The endothelium-independent relaxations to cromakalim, a hyperpolarizing vasodilator acting via ATP-sensitive potassium channels, were also markedly augmented by ethanol feeding in both young and aged rats. In conclusion, ethanol consumption in both young and aged rats was associated with markedly improved arterial relaxations to isoproterenol and cromakalim, as well as clearly augmented relaxation to acetylcholine during inhibition of cyclooxygenase and nitric oxide synthase. These findings suggest that especially the potassium channel-related component of arterial relaxation was augmented by long-term ethanol exposure.

Many studies have suggested that parenteral administration of coenzyme Q 10 (Q 10 ) protects the myocardium of young experimental animals from post-ischemic reperfusion injury. Although parenteral administration, in contrast to per os supplementation, seems to elevate coenzyme Q concentrations in heart tissue, it is not suitable for prophylactic use. In addition, the incidence of ischemic events is greatest in older age. We studied the effect of Q 10 supplementation on myocardial postischemic recovery in 18-month-old Wistar rats. The treated group (n = 9) received 10 mg/kg day of Q 10 for 8 weeks in their chow while the normal chow of the control group (n = 9) contained less than 0.5 mg/kg day of Q 10 . The treatment clearly elevated plasma Q 10 concentration (286 ± 25 μmol l and 48 ± 30 μmol l , treated and controls, respectively, p 9 nor Q 10 concentrations in heart tissue were affected by the supplementation. The isolated perfused hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The preischemic values of developed pressure (DP) but not contractility ( +DP Δt ) and relaxation ( −DP Δt ) were improved by Q 10 supplementation (p = 0.034, p = 0.057 and p = 0.13, respectively) while in postischemic recovery no differences were observed between the groups (p > 0.05 at all time points). Also, in myocardial flow, myocardial oxygen consumption (MVO 2 ) and myocardial aerobic efficiency (DP/MVO 2 ) the groups did not differ at any time points. Although dietary Q 10 supplementation clearly elevated plasma Q 10 concentrations in senescent rats, the coenzyme Q contents in heart tissue and myocardial recovery from ischemia were not affected. However, it is possible that the site of action for the reported beneficial effects of Q 10 is in the coronary endothelium rather than myocardium itself.

1. Age-associated deterioration of arterial function may result from long-lasting oxidative stress. Since coenzyme Q (Q10) has been suggested to protect the vascular endothelium from free radical-induced damage, we investigated the effects of long-term dietary Q10 supplementation on arterial function in senescent Wistar rats. 2. At 16 months of age, 18 rats were divided into two groups. The control group was kept on a standard diet while the other group was supplemented with Q10 (10 mg kg(-1) day(-1)). In addition, nine rats (age 2 months) also ingesting a standard diet were used as the young control group. After 8 study weeks the responses of the mesenteric arterial rings in vitro were examined. 3. Endothelium-independent arterial relaxations to isoprenaline and nitroprusside (SNP) were attenuated in aged rats. Increased dietary Q10 clearly enhanced the relaxation to isoprenaline, but did not affect the response to SNP. In addition, vasodilation of noradrenaline-precontracted rings to acetylcholine (ACh), which was also impaired in aged vessels, was improved after Q10 supplementation. Cyclooxygenase inhibition with diclofenac enhanced the relaxation to ACh only in young rats, while it abolished the difference between the old controls and Q10 supplemented rats, suggesting that the improved endothelium-dependent vasodilation observed in Q10 supplemented rats was largely mediated by prostacyclin (PGI2). 4. In conclusion, long-term Q10 supplementation improved endothelium-dependent vasodilation and enhanced beta-adrenoceptor-mediated arterial relaxation in senescent Wistar rats. The mechanisms underlying the improvement of endothelial function may have included augmented endothelial production of PGI2, increased sensitivity of smooth muscle to PGI2, or both.

Aging and the diseases that typically follow with increasing age, notably atherosclerosis and cancer, are often proposed to be involved in increased oxidative stress. Animal studies, on the other hand, show no clear-cut pattern of age-related changes in enzymatic antioxidant defences. In this study we have demonstrated that total peroxyl radical scavenging antioxidant capacity (TRAP) in human plasma changes with age. We also found that among the antioxidants in human plasma there exists a major fraction of so far unidentified antioxidant(s). A chemiluminescent TRAP assay was used to determine the presence of peroxyl radical scavenging antioxidants in human plasma. The material consisted of 87 healthy volunteers, aged 20-96 years, who used no regular medication, vitamins, or trace elements. In females, total antioxidant capacity increased significantly during the life span. The increase in TRAP was mainly due to unidentified antioxidants. In males, TRAP increased until age 51-74, and then significantly decreased. The decrease observed among males was also due to the sharp decline in the concentration of unidentified antioxidants.

Aging and gender have a strong influence on the functional capacity of the immune system. In general, the immune response in females is stronger than that in males, but there is scant information about the effect of aging on the gender difference in the immune response. To address this question, we performed a transcriptomic analysis of peripheral blood mononuclear cells derived from elderly individuals (nonagenarians, n = 146) and young controls (aged 19–30 years, n = 30). When compared to young controls, we found 339 and 248 genes that were differentially expressed (p1.5 or

Selegiline is a selective and irreversible monoamine B inhibitor with the capacity to increase the level of several antioxidative enzymes in rat brain. It can protect adrenergic neurons against injury induced by neurotoxins such as MPTP, DSP-4 and AF64A in animal studies. In addition, the protective action is not limited to catecholaminergic cells, as selegiline can also minimize the loss of developing motoneurons after axotomy. The aim of this study was to determine whether selegiline can protect peripheral catecholaminergic neurons against the neurotoxic effect of 6-OHDA. This kind of protective effect against 6-OHDA neurotoxicity has not been reported before. Wistar albino male rats aged 4 or 24 months were treated with selegiline or saline solution 1 h before 6-OHDA injection. At 2 weeks after the 6-OHDA injection, the superior cervical ganglia (SCG) and submandibular glands (SMG) were studied using catecholamine histofluorescence and immunohistochemistry for tyrosine hydroxylase (TH). The number of TH-positive cells in the SCG and the length and number of adrenergic nerve fibers in the SMG were quantified. Our findings showed that 6-OHDA caused a reduction of TH immunoreactivity and catecholamine histofluorescence in neuronal somata, as well as a decrease in the number and length of adrenergic nerve fibers in the submandibular gland. Selegiline pretreatment protected SCG neurons and their postganglionic nerve fibers in SMG against these changes in a dose-dependent manner. The mechanism through which selegiline exerts its neuroprotective effect is as yet unknown.

Aging is associated with a profound reduction of the immune capacity (i.e., immunosenescence), which is manifested as increased morbidity and mortality due to infectious diseases in the elderly. The association of cytomegalovirus (CMV) with several aging-associated phenomena has been extensively characterized, e.g., the accumulation of CD8+ nonproliferative, apoptosis-resistant memory cells that have lost the expression of the costimulatory molecule CD28. However, as the CMV seroprevalence is notably high in elderly individuals, the role of CMV-independent changes has been difficult to analyze. To address this question, we performed a transcriptomic analysis (Illumina Human HT12 microarray) of the peripheral blood mononuclear cells (PBMCs) in a cohort of 90-year-old individuals (CMV seronegative, n=6; CMV seropositive, n=140) using the PBMCs of young CMV-seronegative individuals (n=11) as the controls. The cell type distribution (CD3, CD4, CD8, CD28 and CD14) was analyzed using FACS. The data showed that the gene expression profiles of the CMV+ and CMV- nonagenarians were different compared to the CMV- controls. Compared to the CMV- controls, 667 genes showed altered expression in the CMV- nonagenarians, and 559 genes were altered in the CMV+ nonagenarians. Of these, 337 genes were common. An analysis of the canonical pathways revealed that the number of affected pathways was also different (42 in CMV-, 13 in CMV+; of these, 9 were common). Taken together, these results indicate that the CMV-dependent and CMV-independent changes in the aging of the immune system are fundamentally different.

Interleukin-7 is a homeostatic cytokine that contributes to the maintenance of the T cell pool. It also has proinflammatory effects and is involved in the pathogenesis of autoimmune diseases. Due to its homeostatic effects, IL-7 has been proposed as a potential rejuvenation factor for the elderly immune system. We analyzed the correlation of plasma IL-7 concentrations and the proportions of different T cell populations in nonagenarians (n=163) participating in the Vitality 90+ study. Young individuals (n=35, aged 19-30years) were used as controls. The numbers of CD3+, CD14+, CD4+ and CD8+ cells and the expression of the CD28 costimulatory molecule on CD4+ and CD8+ lymphocyte subsets were analyzed using flow cytometry. The plasma IL-7 levels were significantly higher in the nonagenarians compared to the controls (7.86 vs. 5.74pg/ml, p=0.004). In the nonagenarians, plasma IL-7 levels correlated inversely with the proportion of CD3+ T cells and directly with the proportion of CD14+ monocytes and plasma C-reactive protein. No correlation was observed between plasma IL-7 levels and the proportions of CD4+CD28- or CD8+CD28- subsets. These results suggest that the IL-7 levels in nonagenarians do not have an inhibitory effect on the development of immunosenescence; rather they are associated with increased inflammation.

In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7years the number of families with all participating siblings aged 95years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity. In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7years the number of families with all participating siblings aged 95years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.

The effects of aging and chronic ethanol administration on the histochemical and morphometric features of rat superior cervical ganglion were studied in a rat strain selected for voluntary alcohol consumption. Ethanol was administered to the experimental group ad libitum (10% v/v in drinking water) from 3 months to 28 months of age, the average ethanol intake being 6.4–5.4 g/kg per day. The sympathetic neurons of the ethanol consuming rats showed several signs of enhanced degeneration, e.g. decreased neuronal packing density, increased amount of age-pigment and decreased intensity of catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. The results may indicate a selective vulnerability of peripheral sympathetic neurons rather than a universal accelerated aging due to chronic ethanol exposure.

The interactions of aging and long-term voluntary ethanol consumption were studied in the alcohol-preferring AA (Alko Alcohol) rats. The mean daily ethanol intake was 6.45 ± 0.31 g/kg/dy (mean ± SE at the beginning of the exposure at 3 months of age. The control animals were given only food and water ad libitum. There was no difference in survival or weight gain between the control and ethanol groups. When tested for voluntary ethanol intake at the age of 24 months, the rats in the ethanol group consumed significantly more ethanol than the controls. The two groups did not differ in ethanol-induced motor impairment, sleep-time, or hypothermia, nor in the rate of ethanol elimination. The 24-month-old animals, however, showed higher sensitivity to ethanol than the 3–4-month-old rats in the sleep-time test. It is concluded that the feeding regimen used in this study did not produce any detecaable intetactions between ethanol and the aging processes in the AA rats.

Sympathetic ganglion tissue of young (3 months) and aged (24 months) NMRI mice was allotransplanted into the submandibular gland to study the influence of aging on the survival of grafted neurons. The submandibular gland (SMG) was chosen as a host tissue because of its high concentration of NGF and good blood supply. Four weeks postgrafting the viability of transplants was evaluated using the formaldehyde-induced fluorescence technique, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. The density of neurons, catecholamine fluoresence and TH immunoreactivity (THIR) appeared to be almost unchanged when the transplant was completely surrounded by the SMG tissue, whereas transplants located within the interlobular septum and capsule, or even outside the capsule, showed significantly reduced neuronal survival. The shape of most of the transplanted neurons was not different from those in the intact ganglia. The average diameter of the transplanted young neurons was significantly decreased; this was not the case with the aged neurons. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants in both age groups. The transplants in mice treated with 6-OH-dopamine showed considerable regrowth of adrenergic nerve fibers. There seemed to be no marked difference in the survival of transplanted neurons between aged and young animals. The results indicate that the sympathetic neurons from both young adult and aged animals survive the allotransplantation procedure. The neurotrophic factors together with dense vascularization present in the mouse submandibular gland may be beneficial for the restoration of the integrity of mature and aged adrenergic neurons.

Ageing is characterized by chronic low-grade inflammation and the expected lifespan may be affected by several immunological and inflammatory mediators. In this study, we investigated whether the functional Tyr402His polymorphism (rs1061170) on complement factor H (CFH) gene, which is a key inflammatory downregulator, modulates the longevity of 491 nonagenarians in the Vitality 90+ study. Logistic regression analysis and Kaplan–Meier method with the log rank test were used to examine the effect of the CFH Tyr402His polymorphism on 4-year mortality. After follow-up, we observed that risk factor-adjusted mortality was significantly higher among the carriers of CFH 402His allele compared to non-carriers (OR 1.78, 95% CI 1.19–2.67, p = 0.005) and that the survival curves of CFH 402His carriers and non-carriers deviated significantly ( p = 0.016). We propose that the increased mortality is inflammation-related and mediated by aberrant complement regulation by the CFH 402His variant.

In the present study, we combined 2 thoroughly studied markers of inflammation and lipid metabolism—sensitive C-reactive protein (hsCRP) and apolipoprotein E genotype (apoE)—to predict mortality in the elderly (1)(2). Plasma hsCRP concentration (1) and apoE genotype(2) are both important predictors of coronary artery disease (CAD) and stroke (1)(2). The risk of myocardial infarction in people with hsCRP concentrations in the highest third of the population range is twice that of those with hsCRP in the lowest third (1). ApoE genotype is a key regulator of lipoprotein metabolisms (2)(3), and the e4-allele has been associated with increased plasma LDL cholesterol concentration and CAD (2)(3). These 2 factors are biologically linked; low hsCRP concentrations have been measured in apoE e4-allele carriers, indicating that variation in plasma hsCRP concentration is partially determined by the common apoE (e2, e3 and e4) gene polymorphism (3)(4). ApoE e4 allele …

Summary Objective Apolipoprotein E (APOE) genotype is a regulator of hepatic lipoprotein metabolisms and has been linked with longevity. The relationship between APOE genotype and plasma C-reactive protein (CRP), which is produced by the liver during inflammation, has not been studied in nonagenarians. The aim of the present study was to establish whether APOE genotype is related to plasma concentrations of CRP and lipids, or longevity among nonagenarians. Design and patients This cross-sectional study consisted of 291 Finnish nonagenarians and three previously described and genotyped control populations from the same area (i.e. newborns, 40-year-olds, and 70-year-olds). Results In all nonagenarians and especially in women (P= 0.038), CRP level decreased linearly in the genotype order of ɛ2/2, ɛ2/3, ɛ3/3, ɛ2/4, ɛ3/4 and ɛ4/4. Total (P= 0.009) and low-density lipoprotein (LDL) cholesterol (P = 0.076) levels, in turn, were increased in the ɛ4 allele carriers. In newborns, the ɛ4 frequency was 0.192, in 40-year-olds 0.181, in 70-year-olds 0.179 and in nonagenarians 0.095 (P < 0.0001). The decrease in the ɛ4 allele frequency in the elderly was more clearly seen in women than in men. Conclusions APOEɛ4 allele seems to be associated with decreased inflammatory response as measured by CRP among nonagenarians. This finding may partly explain why some ɛ4 allele carriers can reach very old age despite increased risk of hypercholesterolaemia.

Indoleamine 2,3-dioxygenase (IDO), an enzyme degrading tryptophan (trp) to kynurenine (kyn), suppresses T cell activity. Ageing of the immune system, immunosenescence, includes a decline in T cell function. We therefore sought to establish whether IDO activity is involved in immunosenescence and whether it predicts mortality in aged subjects. We measured kyn/trp, reflecting IDO activity, in 284 nonagenarians and 309 blood donor controls. IDO activity was significantly higher in nonagenarians compared with controls and IDO activity at study entry predicted subsequent mortality in nonagenarians. Thus, increased IDO activity might be a mechanism involved in the decline of T cell responses in immunosenescence.

The effects of chronic, intermittent ethanol exposure on brain cytochrome c oxidase (CO) activity levels were studied in young (3- to 4-month-old) and aged (29- to 30-month-old) male Wistar rats. The rats were given highly intoxicating doses of ethanol three times a day by intragastric intubation for four successive days, followed by a 3-day ethanol-withdrawal period. This 4-day ethanol-exposure with 3-day ethanol-withdrawal cycle was repeated five times to simulate the binge drinking of human alcoholics. The histochemical demonstration of CO showed a markedly decreased activity level in the medial prefrontal cortex (especially layer V pyramids and neuropil) of the ethanol-exposed rats of both age groups compared with findings for the respective controls. In the cerebellar vermis, CO activity level was decreased in the Purkinje neurons of the aged ethanol-exposed rats and in the granule cells of both young and aged ethanol-exposed rats. The CO activity level in the locus coeruleus was decreased in both young and old ethanol-exposed rats, but the decrease was more pronounced in the young ethanol-exposed group. Aging per se did not markedly change CO histochemical findings in either prefrontal or cerebellar cortex, but CO activity levels were increased in the locus coeruleus. In summary, results of the current study support our conclusion that CO activity levels were decreased in the cerebral and cerebellar cortices as well as in the locus coeruleus—CNS regions known to be negatively affected by chronic ethanol exposure. Defective energy metabolism due to decreased CO activity levels might compromise neuronal energy stores and thereby contribute to ethanol-induced brain dysfunction and irreversible CNS degeneration.

Aims: The number of cerebellar microglia after 5 1 ⁄ 2 months of continuous or intermittent ethanol exposure was studied using the optical dissector method. Methods: Male Wistar rats were divided into three groups: an intermittently ethanol-exposed group, a continuously ethanol-exposed group and a control group ( n = 6 in each group). The intermittently treated rats had two ethanol- withdrawal periods per week throughout the experiment. The number of microglia was measured in the anterior (folium II) and the posterior (folium X) cerebellar vermis. Tomato ( Lycopersicon esculentum) lectin was used to stain the cerebellar microglia. Results: The volumes of folia II and X were similar in all the groups. The number of microglia increased in the molecular layer of foliu m II in the intermittently ethanol-exposed group compared with the continuously exposed and control groups. In the granular layer, ther e were no differences between the groups in the number of microglia. Conclusions: The results suggest that the number of cerebellar microglia increases in the anterior vermis before any ethanol-induced cerebellar atrophy is discernible. Repeated ethanol withdrawals see m to be more essential in inducing microgliosis than ethanol intoxication per se.

The effects of drinking ethanol throughout a lifetime on voluntary drinking behavior and ethanol-induced motor impairment were studied in alcohol-preferring AA (Alko, Alcohol) and alcohol-avoiding ANA (Alko, Non-Alcohol) rats of both sexes. At the age 3 months, the rats were tested for individual voluntary ethanol (10% vol./vol.) intake and ethanol-induced motor impairment (2 g/kg, i.p.). The rats were housed in group cages, half of them having 12% (vol./vol.) ethanol as the only source of fluid and the other half having free access to water. Food was always available for all animals. At the age of 23 months, their individual voluntary ethanol intake and ethanol-induced motor impairment were tested again. During forced drinking, the females of both strains consumed more ethanol than did the males. The ethanol consumption of the AA and ANA females and the ANA males increased significantly (P.001) with age, but a slight decrease was seen in the ethanol consumption of the AA males. Time x strain interaction showed a significant (P.05) difference in the ethanol consumption of male rats, with the AA males having a slight decrease in ethanol consumption with age, whereas the ANA males increased their ethanol consumption. After 19 months of forced ethanol exposure, AA males significantly decreased their individual voluntary ethanol consumption, and individual voluntary ethanol consumption by ethanol-exposed AA males was more pronounced (P.001) than that of the AA rats that had free access to water (P.05). For the female AA rats, those having free access to water significantly decreased their voluntary ethanol consumption (P.05), but those having ethanol only did not. No significant changes in voluntary ethanol consumption with age or with different exposures were seen in the ANA rats. Body weights were higher in the groups having access to water than in the ethanol-only groups, but the differences were not significant within the AA and ANA strains. The ANA rats were significantly heavier in all groups. These results indicate that the voluntarily nondrinking ANA rats can drink almost as much ethanol as the voluntarily drinking AA rats when they are forced to drink ethanol and that lifelong forced ethanol drinking does not change their inherent drinking habits. When sensitivity to ethanol was measured with the tilting-plane test, the old AA female rats were more sensitive to ethanol than were the young ones. The young ANA females were more sensitive than the AA females when tested at 4 months. In males, aging did not produce any differences in ethanol sensitivity.

Acetaldehyde, the first metabolite of ethanol, has been shown to be capable of binding covalently to liver proteins in vivo, which may be responsible for a variety of toxic effects of ethanol. Acetaldehyde-protein adducts have previously been detected in the liver of patients and experimental animals with alcoholic liver disease. Although a role for acetaldehyde as a possible mediato r of ethanol-induced neurotoxicity has also been previously suggested, the formation of protein-acetaldehyde adducts in brain has not been examined. This study was designed to examine the occurrence of acetaldehyde-protein adducts in rat brain after lifelong ethanol exposure. A total of 27 male rats from the alcohol-preferring (AA) and alcohol-avoiding (ANA) lines were used. Four ANA rats and five AA rats were fed 10-12% (v/v) ethanol for 21 months. Both young (n = 10) and old (n = 8) rats receiving water were used as controls. Samples from frontal cortex, cerebellum and liver were processed for immunohistochemical detection of acetaldehyde adducts. In four (tw o ANA, two AA rats) of the nine ethanol-exposed rats, weak or moderate positive reactions for acetaldehyde adducts could be detec ted both in the frontal cortex and cerebellum, whereas no such immunostaining was found in the remaining five ethanol-treated rats or in the control rats. The positive reaction was localized to the white matter and some large neurons in layers 4 and 5 of the front al cortex, and to the molecular layer of the cerebellum. Interestingly, the strongest positive reactions were found among the ANA rats, wh ich are known to display high acetaldehyde levels during ethanol oxidation. We suggest that acetaldehyde may be involved in ethanol-ind uced neurotoxicity in vivo through formation of adducts with brain proteins and macromolecules.

In this experiment we studied the effects of aging and lifelong ethanol consumption on rat peripheral sympathetic neurons. The aim was to find out the possible differences in the vulnerability to ethanol-induced neuronal degeneration between rats of both genders, or between the alcohol-avoiding (ANA) and the alcohol-preferring (AA) lines of rat. The superior cervical ganglia (SCG) of 40 male and 41 female AA and ANA rats were analyzed. The ethanol-exposed groups had 12% ethanol as the only available fluid from 3 to 24 months of age. The young (3 months) and old (24 months) control groups had water instead. SCG neuronal density, volume, and total neuron number were measured by unbiased morphometric methods. No gender difference was seen in either the volume of the SCG or in the SCG neuron number. The volume of the ganglion was significantly increased with age, but the total neuron number did not change. Neuronal density was significantly decreased with age, but lifelong ethanol consumption induced no further decrease. SCG neuron number in the ethanol-exposed groups did not differ from the age-matched or young control groups, but a significant negative correlation (r = -0.70, p

The effects of lifelong ethanol exposure and aging on the morphology of the locus coeruleus (LC) were studied in the AA (Alko, Alcohol) and ANA (Alko, Nonalcohol) rats of both sexes. The ethanol-consuming (EtOH) rats were given 12% (v/v) ethanol as the only drinking fluid from 4 to 22 months of age, whereas the young (3-month-old) and aged (24-month-old) controls had only water available. The total LC neuron numbers were obtained by using the unbiased disector method. In the AA line, as we have previously reported. the EtOH female and male rats displayed a 26-30% loss of LC neurons compared with the controls. In the ANA line, the EtOH females had 30% fewer LC neurons than the controls (EtOH 1579 +/- 377 vs. controls 2264 +/- 269, ANOVA p0.01), whereas the EtOH males showed no neuron loss compared to the controls (EtOH 1848 +/- 525 vs. controls 2216 +/- 152, ANOVA NS). However, taking into account (sex by line ANCOVA) the markedly higher ethanol intake of the female rats in both lines, no gender or line differences in the ethanol-induced LC degeneration were detected. Neither was there any difference in LC neuron numbers between the young and old control rats of either line of rats. In conclusion, chronic alcohol consumption, not aging per se, damages the LC neurons in experimental animals.

The effects of lifelong ethanol consumption and ageing on the morphology of locus coeruleus (LC) were studied in alcohol-preferring AA (Alko Alcohol) rats of both sexes. Ethanol (12% v/v) was the only available liquid for the ethanol-consuming rats from 3 months up to 24 months of age. Young (3-month-old) and old (24-month-old) control groups were included in the measurements. The LC morphometry was performed by an unbiased disector method. In the old control rats, the total neuron number, neuronal density and the volume of the LC proper did not differ from the young controls. In the ethanol-exposed rats, the total neuron number of the LC was decreased by 30% and the LC neuronal density by 22%, compared to the age-matched controls. No gender difference was found in the vulnerability of LC neurons to ethanol-induced degeneration. The results suggest a remarkable sensitivity of the LC neurons to the ethanol-induced degeneration in both male and female rats. The possible mechanisms and functional implications of this neuronal loss are discussed.

Aging and chronic alcohol consumption can cause degenerative changes in the cerebellar cortex. In this study, the effects of aging and lifelong alcohol consumption on cerebellar cortical layer volumes (molecular and granular) and also white matter layer volumes were studied in alcohol-preferring (AA) and nonpreferring (ANA) rats of both sexes. The ethanol-consuming animals (EtOH) had 12% (w/v) ethanol as the only available fluid from 4 to 22 months of age, whereas the young (3 month) and old controls (24 months) had only water to drink. The volumes of molecular, granular, and white matter layers of the cerebellar vermis in folia II, IV, VII, and X were measured by using systematic sampling and a point-counting method. The volumes of the granular and white matter layers showed consistent increase between 3 and 24 months of age, whereas the volume of the molecular layer remained unchanged with increasing age. Individual ethanol intake was measured over a 1-week period at the beginning and at the end of chronic ethanol exposure. Significant (ANOVA, p = 0.000) sex difference was found in the drinking behavior in both lines, with females consuming more alcohol than males (daily ethanol consumption at 22 months of age 3.2 +/- 0.3 vs. 7.1 +/- 0.3 g/kg for AA males and females; 3.2 +/- 0.3 vs. 5.4 +/- 0.4 g/kg for ANA males and females, respectively). The only ethanol-induced effect on the cerebellum was observed in ANA-EtOH females with a 15% reduction in the volumes of the molecular and granular layer in folium II compared with age-matched controls and a significant (p < 0.05, analysis of covariance with ethanol intake as a covariate) line difference in folium II (molecular and granular layers) was observed between ANA-EtOH females and AA-EtOH females. Furthermore, the volume of the molecular layer in folium II was significantly (p < 0.05, analysis of covariance with ethanol intake and body weights as covariates) reduced for ANA-EtOH females, compared with ANA-EtOH males indicating a sex difference in the cerebellar degeneration due to chronic alcohol consumption. Of the three layers studied, the white matter layer was the most resistant layer to the effects caused by chronic alcohol consumption. In view of the fact that AA and ANA rats of both sexes differ regarding the drinking behavior and ethanol metabolism, they provide an important model for further research on ethanol-induced pathological changes in the central nervous system.

The effects of dexmedetomidine, a selective alpha 2-adrenoceptor agonist, on rat sympathetic neurons were studied during a 12-day, heavy ethanol exposure. Adult male Wistar rats were given ethanol or isocaloric sucrose three times a day by intragastric intubation. Both acute (a single dose of 300 micrograms/kg p.o.) and chronic (100 micrograms/kg x 2 P.O. throughout the experiment) effects of dexmedetomidine were tested. The superior cervical ganglia (SCG) of the ethanol-exposed, non-dexmedetomidine-treated rats showed an abnormally high overall level of tyrosine hydroxylase immunoreactivity (TH-IR) and catecholamine histofluorescence. However, a subpopulation of neurons had apparently lost their catecholamine synthetic activity, as they exhibited no TH-IR or catecholamine fluorescence. The ethanol-exposed ganglia also showed structural alterations (e.g., decreased neuronal size and increased occurrence of vacuolated neurons). In the ethanol-exposed, chronically dexmedetomidine-treated group, by contrast, the SCG exhibited TH-IR and catecholamine fluorescence intensities comparable to those seen in the control ganglia. All the structural parameters studied, as well, were at the control level in the chronically dexmedetomidine-treated group. The single dose of dexmedetomidine offered only marginal protection against the ethanol-induced alterations. These results suggest that chronic dexmedetomidine treatment may prevent ethanol-induced overactivity and degeneration of catecholaminergic neurons.

Age-differences in the sensitivity of peripheral sympathetic neurons to chronic ethanol exposure and ethanol withdrawal were studied in male Wistar rats aged 4 months, 12 months, or 24 to 25 months. The superior cervical ganglia (SCG) of the young (4 months) and the 2-year-old rats responded to a 12-day or 4-week ethanol exposure with significantly increased catecholamine turnover, while the ganglia of the middle-aged rats (12 months) showed only a minor increase in the intensity of catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. Extensive neuronal vacuolation was found in the 4 months ethanol-exposed SCG, probably as a reaction of a subpopulation of neurons to increased stimulation. Ethanol-induced neuronal loss was most prominent in the SCG of the oldest age group. Contrary to the marked changes in SCG functional and morphometric parameters, the pelvic sympathetic neurons in the hypogastric ganglion showed no significant changes after ethanol exposure. The pattern of ethanol-induced morphological alterations found in the present study did not provide unambiquous support for either the "accelerated aging" or the "increased vulnerability" concept regarding ethanol-aging interactions in the nervous system.

The myocardial interactions of ageing and lifelong ethanol ingestion were studied in the ethanol-preferring AA (Alko Alcohol) line of rats. Samples of the left ventricle from young control rats (3-month), old control rats (30-month) and rats exposed to ethanol from 3 to 30 months of age, were studied in terms of myocardial ultrastructure and lipopigmentation. Electron microscopic morphometry showed an age-related increase in the volumetric densities of lipofuscin and unspecified sarcoplasm, while the proportions of mitochondria, myofibrils and tubular structures remained unaltered in the left ventricular myocardium. Lifelong ethanol exposure increased the proportion of sarcoplasmic reticulum and transverse tubules, apparently due to dilation of the tubular structures. Mitochondria were significantly larger in the ethanol-exposed rats compared to the control rats of the same age, while the volumetric proportion of mitochondria tended to decrease in the ethanol-exposed group. Fluorescence microscopic image analysis showed that myocardial lipopigmentation (proportion of myocardial area covered by autofluorescent lipopigrnents) was about two-fold in the old ethanol-exposed rats compared to the old control rats, and 13-fold compared to the young controls. It is concluded that ageing and chronic ethanol ingestion produce rather different patterns of alteration in myocardial ultrastructure, which does not support the concept of ethanol-induced accelerated ageing. The enhancement of myocardial lipofuscin accumulation is thought to reflect chronic oxidative stress in the hearts exposed to ethanol.

The effect of chronic (3-4 weeks), heavy ethanol exposure on neuronal vacuolation in rat peripheral nervous system was studied in male Wistar rats. The rats were force-fed with 25% ethanol 3 times a day, which resulted in blood ethanol levels of 53.1 +/- 18.8 mmol/l, i.e., marked intoxication. In the superior cervical ganglia (SCG), the ethanol exposure increased the proportion of vacuolated neurons c. 13-fold (0.2 +/- 0.0% in the control ganglia, 2.7 +/- 0.6% in the EtOH-ganglia, P < 0.001). A considerable population of vacuolated neurons (VN) was seen in the sensory inferior vagal (nodose) ganglia, and occasional neurons with large cytoplasmic vacuoles in the sensory dorsal root ganglia (DRG) of the EtOH-rats. In the hypogastric ganglia, where VN are regularly found in the adult rat, ethanol exposure did not affect the amount or the appearance of the vacuolated neurons. The number of VN in the SCG decreased significantly between 2 days and 1 week after cessation of the exposure, but did not return to control level by 1 month after ethanol withdrawal. In electron microscopy, most of the vacuolated SCG neurons showed normal ultrastructure, apart from the large cytoplasmic vacuoles. Some vacuolated neurons, however, showed neuropathologic changes, e.g., dilated endoplasmic reticulum, mitochondrial alterations and increased numbers of myelin figures. These degenerative changes were more frequent in the vacuolated DRG neurons than in the sympathetic ones. The occurrence of VN in rat peripheral ganglia may represent a reaction to increased stimulation during prolonged ethanol exposure and, especially, during repeated phases of ethanol withdrawal.

Sympathetic ganglion tissue of aged (36 months) Wistar rats was allotransplanted into the submandibular gland (SMG) of young (3 months) animals to study whether sympathetic neurons can outlive the original host. The visbility of the transplants was evaluated one yeat postgrafting, using the formaldehyde-induced fluorescence technique (FIF) for histochemical demonstration of catecholamines, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. One year after transplantation, grafted superior cervical ganglion (SCG) cells demonstrated catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. The transplants consisted of groups of sympathetic neurons dispersed in a fibrous matrix. After long postgrafting time, the sympathetic neurons of aged rats showed several signs of enhanced degeneration; increased autofluoroscent lipopigment, decreased neuronal density and reduced catecholamine fluorescence. The mean diameter of the transplanted aged neurons was significantly decreased. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants. A subpopulation of small and medium-sized grafted neurons sent out fluorescent fibers, which were located in a fibrous scar area but did not extend into submandibular host tissue. The results indicate that a long postgrafting time induced degeneration which is comparable to normal aging changes in grafted very old neurons. Thus, aged sympathetic neurons maintain plasticity to survive as transplants, and under favourable conditions these neurons outlive the original host.

The dependence of superior cervical ganglion (SCG) adrenergic neurons on their target organ submandibular salivary gland containing high concentrations of nerve growth factor was studied in adult and aged male mice. The submandibular salivary glands were removed (sialectomy) either uni- or bilaterally, and the SCG were studied by fluorescence microscopy and histochemically. Catecholamine fluorescence and tyrosine hydroxylase immunoreactivity decreased after sialectomy, suggesting reduced noradrenaline production. Neuronal density was lower in the aged controls than in the young controls. In both age groups, sialectomy reduced the density of catecholamine-producing neurons. In the mouse SCG, there was remarkable heterogeneity in the size of neuronal somata. In aged control mice there was a greater number of large-size neurons than in young adult control mice. Six weeks postoperatively, no large catecholamine-producing neurons could be observed in the ganglia. Yellow autofluorescent lipopigments accumulated with age in the adrenergic neurons. Sialectomy increased the accumulation of lipopigments in both young and aged neurons. Sialectomy resulted in (a) reduced catecholamine fluorescence, (b) reduced tyrosine hydroxylase immunoreactivity, (c) reduced number of catecholamine neurons, (d) increased autofluorescent lipopigment. Ageing resulted in (a) reduced number of neurons, (b) increased ratio of large to small neurons, (c) increased autofluorescent lipopigment. Alterations after sialectomy were more detrimental in the aged ganglia than in the young adult ganglia. The discontinuation of the retrograde supply of nerve growth factor may contribute to these alterations.

The localization of PKC-beta was studied in rat sympathetic neurons using a polyclonal antibody specific for the beta 1- and beta 2-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC-beta-LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC-beta-LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC-beta-immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC-beta-LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC-beta in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC-beta in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.

Adrenal medullary tissue was autotransplanted to the superior cervical ganglion (SCG) of aged (26 months old) and young adult (3 months old) rats. Four and 20 weeks after operation, the viability of the transplants was evaluated using the formaldehyde-induced fluorescence (FIF) technique and tyrosine hydroxylase (TH) immunocytochemistry. Four weeks postgrafting, the transplant consisted of a densely-packed group of intensively fluorescent chromaffin cells in both age groups. The cells showed strong TH immunoreactivity and some of them were elongated, but only a few displayed short processes. At 20 weeks, most of the cells were spindle shaped and sent out fluorescent processes and a few of them were transformed toward ganglion-like cells. The results suggest that both young and old adrenal chromaffin cells are able to survive, produce neuronal processes and transform toward a neuronal phenotype in the rat superior cervical ganglion.

The accumulation of lipopigments during aging in several peripheral organs and in the nervous system is considered to be related to the peroxidation of unsaturated fatty acids. In this study the effect of lifelong (until to 18 months) dietary antioxidants selenium and vitamin-E on pigment accumulation in some peripheral tissues was estimated using fluorescence and electron microscopy. In the vitamin E deficiency group, there was increased pigment accumulation in all peripheral tissues studied except the hypogastric ganglion, where no change was observed. The vitamin E supplementation degreased the pigment accumulation in older animals in some of the tissues studied. At the electron microscopical level the accumulated pigment in the adrenal cortex showed a lipofuscin-like structure. Lifelong selenium supplementation or deficiency did not significantly alter pigment accumulation in any of the tissues studied. It is possible that in many organs dietary selenium may not play a critical role in lipofuscin formation.

Sympathetic ganglion tissue of 3-months- and 18-months-old Fischer-344 rats was autotransplanted into the adrenal gland in order to determine the effect of aging on the survival of grafted neurons. Adrenal cortex was chosen as the host tissue because it is well vascularized and has a high concentration of glucocorticoids, which stimulate the synthesis of catecholamines. At 4 weeks following the transplantation, the density of neurons was decreased in all transplants, but approximately the same proportion of remaining neurons showed tyrosine hydroxylase immunoreactivity as in intact ganglia. At 8 weeks, a subpopulation of large neurons showed an increased accumulation of age pigment. The heavily pigmented neurons were usually devoid of catecholamines, whereas small non-pigmented neurons frequently showed strong catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. There was no marked difference between old and young animals in the survival of transplanted neurons. The results show that the sympathetic neurons from both 3-months-and 18-months-old animals survived the autotransplantation procedure. The humoral environment of the adrenal cortex may be beneficial for the restoration of the integrity of sympathetic neurons.

Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%-80% of which were heavily labelled. About 5%-10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%-70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells. The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.