Senescent Changes Research Articles

Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.

Heat-induced Leaf Senescence and Hormonal Changes for Thermal Bentgrass and Turf-type Bentgrass Species Differing in Heat Tolerance

Leaf senescence can be induced by many environmental stresses, including supraoptimal temperatures. The objectives of this study were to evaluate leaf senescence induced by heat stress for two Agrostis species contrasting in heat tolerance and to examine whether heat-induced leaf senescence in both species was associated with changes in three major senescence-related hormones: ethylene, abscisic acid (ABA), and cytokinins. Plants of heat-tolerant rough bentgrass (Agrostis scabra Willd.) and heat-sensitive creeping bentgrass (Agrostis stolonifera L.) were exposed to 35/30 °C (day/night) (high temperature) or 20/15 °C (control) for 35 d in growth chambers. Turf quality, photochemical efficiency (Fv/Fm), and the contents of two pigments (chlorophyll and carotenoid) for both species decreased under high temperature; however, heat-tolerant A. scabra exhibited delayed and less severe decline in all parameters compared with heat-sensitive A. stolonifera. Ethylene production rate increased in both species at 35 °C, but the increase was observed 21 days later in A. scabra compared with that in A. stolonifera. ABA content increased at the initiation of heat stress and then declined in both species after prolonged heat stress. However, the timing of the increase was delayed for 7 days and the highest level of ABA content was less in A. scabra (4.0 times that of the control) than that in A. stolonifera (5.9 times that of the control). Decreases in both forms of cytokinins (transzeatin/zeatin riboside and isopentenyl adenosine) were also delayed for 14 days and less pronounced in A. scabra. Correlation analysis revealed that leaf senescence induced by heat stress was negatively correlated to ethylene and ABA accumulation and positively correlated to cytokinin production. Delayed leaf senescence in A. scabra under heat stress could be related to slower and less magnitude of changes in ethylene, ABA, and cytokinins.

Variation in senescent-dependent lung changes in inbred mouse strains

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.

Coronary Artery Aneurysm Induced by Kawasaki Disease in Children Show Features Typical Senescence

Kawasaki disease (KD) causes coronary artery disease (CAD) in children. In addition, a history of KD is suspected to be a risk factor for the development of atherosclerotic heart disease in the future. Histological senescence changes are a common denominator in atherosclerotic lesions in adults, so the present study investigated whether histological senescence changes had already occurred in KD aneurysm. KD coronary aneurysms and internal mammary arteries retrieved from 5 children with KD (3, 4, 5, 6, and 11 years old, respectively) who underwent coronary artery bypass grafting, as well as giant coronary aneurysm size-reducing operations, were analyzed. Senescence-associated strong beta-galactosidase activity was observed in KD aneurysms, but not in the internal mammary arteries. An immunohistochemical analysis of the KD aneurysm using anti-CD31, anti-endothelial nitric oxide synthetase (eNOS), anti-vascular adhesion molecule-1 (VCAM-1), and anti-monocyte chemoattractant protein-1 (MCP-1) showed vascular endothelium CD31 staining, decreased staining of eNOS and strong staining of MCP-1 and VCAM-1. cDNA microarray gene expression profiling revealed increased MCP-1 expression in the KD aneurysm, a finding confirmed by quantitative polymerase chain reaction. Histological features of senescence and active remodeling gene expression show that the KD aneurysm is not a silent vasculitis terminal. The future fate of KD aneurysms, including atherosclerosis, should be monitored carefully.

Within-person trial-to-trial variability precedes and predicts cognitive decline in old and very old age: Longitudinal data from the Berlin Aging Study

Neurocomputational modeling and empirical evidence suggest that losses in neuronal signaling fidelity cause senescent changes in behavior. We applied structural equation modeling to five-occasion 13-year longitudinal data from the Berlin Aging Study (n=447; age range at t1=70–102 years) to test whether trial-to-trial reaction time variability in perceptual speed (identical pictures) antecedes and signals longitudinal decline in levels of performance on perceptual speed (digit letter and identical pictures) and ideational fluency (category fluency). Higher trial-to-trial variability preceded and predicted greater cognitive decline in perceptual speed and ideational fluency. We conclude that trial-to-trial variability signals impending decline in cognitive performance, and that theories of neurocognitive aging need to postulate developmental cascades between senescent changes in variability and central tendency.

Mechanism of radiation induced premature senescence of bone marrow stromal cells: experiment with murine bone marrow stromal cells

To investigate the mechanism of radiation induced premature senescence of bone marrow stromal cells induced by gamma-radiation. Bone marrow stromal cells were isolated from the bones of mice, cultured, and divided into 2 groups: control group and irradiation group. The cells of the irradiation group were exposed to 60Co gamma-rays of the dose of 8.0 Gy. 24 h, 72 h, 1 week, and 2 weeks after irradiation MTT method was used to test the proliferation of the cells, Giemsa staining was used to observe the morphology, the percentage of cells positive in beta-galactosidase (SA-beta-Gal), a senescence associated biomarker, was detected by cytochemistry, and RT-PCR was used to detect the mRNA expression of p21(Cip1/Waf1) and p53 gene, both associated with senescence. One week after the irradiation flow cytometry was used to analyze the cell cycle distribution. Characteristics of mature senescence were seen in the cells of the irradiation group. One week after the irradiation the percentage of G0/G1 of the irradiation group was 89.83% +/- 0.05%, significantly higher than that of the control group (66.95% +/- 0.36%, P < 0.01). 24 h, 72 h, 1 week, and 2 weeks after irradiation the percentages of SA-beta-gal positive cells in the irradiation group were 20.33% +/- 0.03%, 34.33% +/- 0.03%, 86.33% +/- 0.02%, and 95.67% +/- 0.02% respectively, significantly higher than those of the control group (2.33% +/- 0.006%, 14.33% +/- 0.02%, 24.67% +/- 0.02%, and 45.00% +/- 0.02% respectively, all P < 0.01). The mRNA expression of p53 and p21(Cip1/Waf1) mRNA expressions in the irradiation group increased 24 h after the irradiation and lasted for two weeks. Gamma-radiation induces changes of premature senescence in bone marrow stromal cells in which p53-p21(Cip1/Waf1) manner pathways may play an important role.

Expression of the beta-amyloid transporter, LRP-1, in aging choroid plexus: implications for the CSF-brain system in NPH and Alzheimer's disease

Background The CSF system, including the choroid plexus, is an important route for removing the A-Beta peptides that, in high concentrations, injure neurons. Central retention of A-Beta peptides occurs when the blood-CSF and bloodbrain barriers suffer deficits in aging and disease. LRP-1, a transporter, which actively removes A-Beta from extracellular fluid, undergoes expression changes in senescence, which is a risk factor for NPH and AD. We hypothesize that LRP-1 activity in the 'barriers' is essential for the wellbeing of the brain's interstitial and cerebrospinal fluids. This study has assessed the expression of LRP-1 in the choroid plexus and cerebral capillaries of aging rats in order to elucidate the state of this A-Beta clearance transporter in senescence. Faulty A-Beta disposal from the CNS may predispose to NPH and the reduced CSF turnover rate in AD.

Aging of the brain, entropy, and Alzheimer disease

Sporadic Alzheimer disease (AD) is related to advancing age far more than to any other risk factor and ultimately affects almost half of the population over age 85. Despite its remarkable prevalence among the elderly, it has been regarded as a specific disease, distinct from "normal aging." This view is supported in large part by clinical and pathologic similarities to early-onset, dominantly inherited familial AD, where genetic mutations related to beta-amyloid have been identified. There is much evidence that sporadic AD overlaps with normal aging in many clinical and pathologic features. Some of the many molecular age-related changes (ARCs) affecting the brain, both intrinsic (programmed) and extrinsic (stochastic), are reviewed, with discussion of the effects they have singly and collectively on neuronal viability and vulnerability. The effect of ARCs on the brain is seen as the biologic manifestation of increasing entropy, an approach that helps to explain the progressive decline of neural and cognitive function over time; the ability of multiple, varied ARCs to summate as individuals age; the transitional relationship between normal aging, mild cognitive impairment, and AD; and the apparent differences between normal aging and AD. Increasing entropy, manifest through a complex network of interacting ARCs, is seen as the fundamental driving cause of neural and cognitive decline in the elderly, as well as the overriding etiologic principle in further transition to sporadic AD. Research on sporadic AD has largely focused on finding a single causal metabolic disorder or genetic mutation. Multiple ARCs contribute to declining function and increased frailty in the aging brain, however, and to the catastrophic disintegration of sporadic AD. Effective prevention or treatment will depend on recognizing the contributions of a multiplicity of ARCs to AD and reducing the burden of as many as possible. The role of amyloid is seen as one element in the larger network of senescent changes involving the aging brain.

Temporal integration of the contralateral acoustic-reflex threshold and its age-related changes

Although numerous studies have investigated temporal integration of the acoustic-reflex threshold (ART), research is lacking on the effect of age on temporal integration of the ART. Therefore the effect of age on temporal integration of the ART was investigated for a broad-band noise (BBN) activator. Subjects consisted of two groups of adults with normal-hearing sensitivity: one group of 20 young adults (ten males and ten females, ages 18-29 years, with a mean age of 24 years) and one group of 20 older adults (ten males and ten females, ages 59-75 years, with a mean age of 67.5 years). Activating stimulus durations were 12, 25, 50, 100, 200, 300, 500, and 1000 ms. Significant main effects for duration and age were obtained. That is, as the duration increased, the acoustic reflex threshold for BBN decreased. The interactions of duration x age group and duration x hearing level were not significant. The result of pair-wise analysis indicated statistically significant differences between the two age groups at durations of 20 ms and longer. The observed age effect on temporal integration of the ART for the BBN activator is interpreted in relation to senescent changes in the auditory system.

Depletion of resting zone chondrocytes during growth plate senescence

With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.

老年医学における未病―老年疾患の予防をどのようにすすめるか―６．老化予防に向けての栄養療法

Overeating and obesity are major health problems even in the elderly since they lead to the metabolic syndrome, resulting in an increase in cardiovascular disease. The development of novel nutritional therapeutics for the purpose of promoting health and controlling ageing process aims at the self-helping elderly. Caloric restriction (CR) has been widely investigated as a powerful method that can prevent and reverse senescent changes. CR might counteract the deleterious aspects of metabolic syndrome and may prolong lifespan even in humans. However, we should keep in mind that several fundamental issues about CR still remain unsolved. In addition, malnutrition and nutritional troubles are emerging problems in the elderly in care. Thus, we emphasize that nutritional intervention aiming at ageing control should be established and be developed as a custom-made therapeutics for the elderly, who show great individual variation.

Aging, Disability, and Frailty: Implications for Universal Design

Throughout the world all populations are seeing burgeoning numbers of "elders", defined as persons aged 65 year and older. In many countries, including Japan, the United States, Norway, Sweden and the United Kingdom, those aged over 65 are at or approaching 15% of the population. As their numbers have increased, so have their health care expenses, leading to extensive research on the health, well being, and life expectancy of these increasingly older elders. Today this group is further sub-divided: the young-old ages 65-74, the old-old ages 75-84, and the oldest-old ages 85+, for both health care and research purposes. However broad variation still characterizes even these groupings. Rates of frailty and disability increase with increasing age among these elders. For example, inabilities to complete at least one activity of daily living increased from about 5-7% at ages 65-69 years to about 28-36% at ages 85+ in 1987. Death rates continue to decline at all ages past 50 years and rates of disability seem to be doing the same. For the foreseeable future, we may expect increasing numbers of older, frail elders than in previous decades. Thus, people are not only living longer, they generally are healthier at advanced ages than were previous cohorts, thus "old age" disabilities of the 20th century will be put off to even older ages during the 21st century. As yet there is no clear way to assess senescent changes in humans, although activities of daily living, allostatic load, and frailty indices have all been suggested. One future need is greater development and use of universal and accessible design in all aspects of the built environment.

Injury prevention and rehabilitation for active older adults

With the rise in active participation in sports and exercise by older people, Injury Prevention and Rehabilitation for Active Older is both timely and instructive. It explores the issues involved in working with active older adults, providing a valuable resource to help sports medicine professionals prevent, diagnose, and treat injuries for this growing population.Geared toward those working with active seniors--from competitive and recreational athletes to fitness enthusiasts--Injury Prevention and Rehabilitation for Active Older Adults contains the information to help practitioners -strengthen their understanding of general issues in sports medicine for active seniors;-explore prevention of, and determine treatment for, specific injuries;-apply to their own practice the knowledge of specialists experienced in working with older populations; and-implement and supervise appropriate conservative therapies.Injury Prevention and Rehabilitation for Active Older was written by a team of specialists with extensive experience in treating active seniors. While it emphasizes conservative treatment over surgery, it also guides readers in knowing when to refer a client to a surgeon, how to prepare a client for what might happen when referred, and what type of surgery might be indicated. Therapists and trainers will strengthen their ability to explain their basis for both treatment and referral. Part I focuses on a variety of issues in sports medicine for active seniors, including senescent changes in the musculoskeletal system, exercise testing and prescription, and factoring the kinetic chain into prevention and therapy. Flexibility, stretching, and massage for older people are also covered, as are nutrition, nutritional supplements, and pharmacology.Part II, organized by anatomical areas, delves into specific injuries and conditions in active seniors. This approach helps readers easily locate regional musculoskeletal problems and identify appropriate rehabilitation procedures. These regions include the shoulder, elbow, hand and wrist, spine, hip, knee, and foot and ankle. Common injuries, conditions, and treatments are explored in each area.Accompanying photos and illustrations supplement the text, showing stretches for all parts of the body, exercises for both injury prevention and rehabilitation, diagnostic techniques (including special tests and best X-ray positions) and various treatment options. The result is a reference that facilitates understanding of the issues involved in preventing and treating injuries in active older people and in helping them recover and return to full activity as soon as possible.

Aging Neuromodulation Impairs Associative Binding

Relative to young adults, older adults are particularly impaired in episodic memory tasks requiring associative binding of separate components into compound episodes, such as tasks requiring item-context and item-item binding. This associative-binding deficit has been attributed to senescent changes in frontal-hippocampal circuitry but has not been formally linked to impaired neuromodulation involving this circuitry. Previous neurocomputational work showed that impaired neuromodulation could result in less distinct neurocognitive representations. Here we extend this computational principle to simulate aging-related deficits in associative binding. As expected, networks with simulated deficiency in neuromodulation resulted in less distinct internal representations than did networks simulating the processing and performance of young adults, and were also more impaired under task conditions that required associative binding. The findings suggest that senescent changes in neuromodulatory mechanisms may play a basic role in aging-related impairment in associative binding by reducing the efficacy of distributed conjunctive coding.

Increased Expression of Senescence-Associated Cell Cycle Inhibitor p16INK4a in Deteriorating Renal Transplants and Diseased Native Kidney

Some features of kidney transplants with dysfunction overlap the lesions of aging, such as tubular atrophy and interstitial fibrosis (TA/IF) without major glomerular abnormalities. Somatic cell limitations could contribute to deterioration in aging and disease states. Since expression of p16(INK4a), a cell cycle inhibitor associated with somatic cell senescence in vitro, is induced in aged kidney, we studied whether kidneys with dysfunction and TA/IF manifested increased p16(INK4a) expression. We performed p16(INK4a) immunostaining on transplanted kidneys and native kidneys with chronic renal diseases. At implantation, transplants manifested little TA/IF, and nuclear p16(INK4a) immunostaining was consistent with age. However, transplants biopsied for abnormal function displaying TA/IF showed strong nuclear and cytoplasmic p16(INK4a) staining, beyond the amount predicted for age. Both atrophic and non-atrophic nephrons displayed increased p16(INK4a), suggesting that it was not simply a feature of atrophy. Epithelial p16(INK4a) staining was not increased in transplants with good function, but was increased in diseased native kidneys. The finding of increased p16(INK4a) expression in renal transplants and diseased kidneys with TA/IF and impaired function supports the concept that some cell senescence changes that accompany aging are also induced by injury and disease stresses. Thus, aging, injury and disease may share common pathways involving somatic cell senescence.

Effect of Maturity at Harvest in Relation to Changes in Antioxidant Properties and Ethylene in ‘Chandler’ Strawberry Fruit

Abstract The relationship between fruit maturation and accumulation of hydrogen peroxide (H202), lipid peroxidation, ethylene (C2H4) production, antioxidant activity (hydrophilic, lipophilic and total) and the antioxidant enzyme ascorbate peroxidase (APX, EC 1.11.1.11) in fruit pericarp tissue of ‘Chandler’ (Fragaria xananassa Duch.) strawberry were measured. ‘Chandler’ fruit pericarp maturation and ripening were accompanied by a decline in H202 content early in fruit development followed by a rapid accumulation. An increase in membrane lipid peroxidation (thiobarbituric acid reactive substances, TBARS) coincided with accumulation of H202, which preceded a rise in C2H4 production. In general, antioxidant activity declined as fruit matured and ripened. APX enzyme activity increased by 2-fold and peaked at the pink stage of development and then gradually declined with ripening. H202 may serve as a signal molecule to initiate the cascade of oxidative processes during maturation and ripening. APX enzyme activity during maturation and ripening was not substantial and thus, may not have a role in alleviating accumulation of H202 and subsequent events related to oxidative senescence in fruit pericarp. To our knowledge, this is the first study to present fractionated antioxidant activities (HAA, LAA and TAA) from strawberry pericarp as assessed by the ABTS*+ radical cation assay. A fundamental understanding of the mechanisms involved in the senescent related-oxidative changes during strawberry fruit ontogeny in relation to quality and nutrition is discussed.

Expression of p16INK4A variants in senescent human fibroblasts independent of protein phosphorylation

Upregulation of the p16 tumor suppressor is a hallmark of senescence in human fibroblasts. In this study, we investigated potential protein modification of p16 in senescent human fibroblasts using 2D SDS-PAGE analysis. Three distinct p16 variants with isoelectric points of 5.2, 5.4, and 5.6, were consistently detected in normal human IMR90 fibroblasts that had undergone senescence due to forced expression of oncogenic H-ras or culture passage. Moreover, in contrast to short-term serum starvation, which induces quiescence, IMR90 fibroblasts cultured in low serum for a prolonged period exhibited senescent phenotypes and expression of the three p16 variants. All three p16 variants are unlikely phosphoproteins since they failed to react with antibodies against phospho-serine, and were resistant to the treatment with phosphatases. Functionally, co-immunoprecipitation assays using antibodies against cdk4 and/or cdk6 revealed that only the two most acidic p16 variants associated with cdk4/6. Moreover, senescence induced by the forced expression of p16 in early passage IMR90 fibroblasts or osteosarcoma U2OS cells was accompanied by expression of the two most acidic p16 variants, which also associated with cdk4/6. In summary, we report that prolonged serum starvation-induced senescence may provide an additional model for studying biochemical changes in senescence, including p16 regulation. Furthermore, induction of endogenous p16 in senescent human fibroblasts correlates with the expression of three distinct p16 variants independent of protein phosphorylation. Lastly, expression of the two cdk-bound variants is sufficient to induce senescence in human cells.

Senescence of spatial chromatic contrast sensitivity II Matching under natural viewing conditions

Age-related changes in the spatial chromatic contrast sensitivity function of detection, measured along S and L - M cone axes, were demonstrated in a companion paper [Hardy et al., J. Opt. Soc. Am. A 22, 49 (2005)]. Here senescent changes in chromatic contrast appearance were assessed by contrast-matching functions (CMFs). Luminance and chromatic CMFs (S and L - M axes) were compared for younger (age 18-31 yr) and older (age 65-75 yr) trichromatic subjects by using stimuli that were perceptually anchored to the same physical standard contrasts. Subjects matched the contrast of test gratings of various spatial frequencies (0.5-8 cycles per degree) to the standard stimuli under natural viewing conditions. Because of changes in the visual system with age, the standard stimuli were closer to threshold for older subjects; however, in general, the shapes of the CMFs were similar for both groups. The results suggest that the perception of relative contrasts across spatial frequencies is stable with age.

Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.