Effects Of Calorie Restriction On The Behavior And Hippocampal Histology And SirT1 And FoxO3a Signaling Pathway Of Early Old Age Rats

Background and objectiveCalorie Restriction (CR) is one of the most effective methods to increase longevity,delay ageing and improve health. CR retards age-related neurondegeneration, which ischaracterized by decline in learning and memory ability. Majority of former data wereobtained from life-long CR rats or mice. Whereas there are numerous reports aboutage-related diseases prevention by CR, the effects of CR on cognitive performanceare largely unknown. We assumed that the maintained learning and memory ability inCR rat was related to the postponed neural senescence in hippocampus, where is oneof the most important regions involved in learning and memory. As a senescencemarker, senescence associatedβ-galactosidase (SA-β-GAL) has been widely adoptedin replicative senescence in vitro; however, the reliability of this biomarker in braintissue has not been well understood. In this investigation, we applied a shorter periodof the treatment of CR from early old age rats and evaluated the survival and learningand memory ability by behavioral study. We applied SA-β-GAL to detect senescentneurons in hippocampus to examine the senescence status of hippocampal neurons ofCR rat. Meanwhile, we investigated the activity of SA-β-GAL in rat hippocampus atdifferent ages and in prolong cultured hippocampal neurons, as a parallel study to testthe marker's reliability.Materials and methodsMale Sprague-Dawley (SD) rats with age of 18 month were subjected to restrictedintake in CR group (n=29) by 60％comparing with that of rats fed ad libitum (AL)(n=31) for 6 months. We compared the overall health status, including survival andlocomotor activity by open-field test. Spatial learning and reference memory ability ofthe rats were evaluated by Morris Water Maze (MWM). Cell numbers in CA3 region of hippocampus were counted after Nissl staining. Hippocampus of 6 Mon (n=10), 18Mon (n=6) and 24 Mon (n=7) SD rats were observed by histochemical staining forSA-13-GAL. Prolonged cultured primary hippocampal neurons of SD rat wereobserved by cytochemical staining for SA-13-GAL.ResultsGrowth rate of the CR rats demonstrated a period of decline during the first 10weeks of treatment, and thereafter became steady below that of AL rats (p＜0.001).The CR rats showed a higher survival (p=0.039). In the open-field test, the CR ratstraveled longer (p=0.021) especially at the centre place (p=0.048), and moved faster(p=0.021) as well as in circumjacent area (p=0.012) than the AL rats. The meanescape latency (EL) of CR group was shorter than that ofAL group (p=0.038) innavigation trials of MWM. The differences in the times crossing the former annualand percentage of time and distance covered in the target quarterⅢdidn't reach thestatistic significance(p=0.324 and p=0.367), however, great difference showed in thatof quarterⅡ(p=0.027and p=0.029). In the histological study, CR rats showed tighterpyramidal cells line and higher neuron number in the CA3 region in hippocampusthan that of AL group in Nissl staining (p=0.039). The staining of SA-β-GAL wasweaker in pyramidal cells of hippocampus of CR rats than in AL rats (p＜0.001).SA-β-GAL activity elevated in hippocampal pyramidal cells of CA3 region withadvancing age (p＜0.001) and with culture period of hippocampal neurons (p＜0.001).Conclusion:1. The 6-month treatment of 60％CR elevated survival of early old age rat.2. The 6-month treatment of 60％CR benefited health status by locomotor activity.3. The 6-month treatment of 60％Calorie Restriction delayed the age-relatedinability to learn and improved the accuracy of the memory of early old rats.4. A Calorie Restriction regimen for early old rats delayed the senescence ofhippocampal neurons may contribute to the better learning and memoryperformance.5. SA-β-GAL was a reliable biomarker to detect senescent cells in CA3 region of hippocampus. Background and Objective:It has been known that calorie restriction (CR) can extends median and maximallife span in a wide spectrum of organisms and retard or prevent age-dependentdeterioration of brain. However the molecular mechanisms of the benefit of CR arestill largely unknown. An understanding of the molecular mechanisms that underliethe beneficial effects of CR will help identify novel dietary, and lifestyle strategies forslowing the rate of aging and preventing these diseases.Studies show that the elevation of silence information regulator 2 (Sir2), anicotinamide adenine dinucleotide (NAD)-dependent deacetylase is one of thecommon effects of CR in a wide spectrum of organisms. It has been postulated thatthe mammalian homolog sirtuin 1 (SirT1) is one of the key regulators of thedownstream effects of CR. SifT1 posses a large list of substrates, including p53,NF-kappaB and forkhead transcription factors (FoxO3a), which modulate cellularresistance to oxidative and genotoxic stress.As one of the members in mammalian FOXO families, FoxO3a is involved incontrolling various biological functions, such as cell cycle arrest, repair of damagedDNA, and senescence. FOXO family is one of the main down-steam targets ofinsulin/IGF-1 pathway, which is another way for CR to extend life-span. In mammals,growth hormone (GH) and insulin-like growth factor 1 (IGF-1) appear to be critical indetermining lifespan. FoxO3a is highly expressed in brain especially in thedeveloping and adult hippocampus, indicating FoxO3a might take a role in memoryformation and modulate neuronal senescence.Both SirT1 and FoxO3a mediate the important pathway for neuronal growth and physiology. However their modulations in CR rats are still unclear. To elucidate thecontribution of both factors to the effects of CR is helpful to understand theunderlining mechanism. We studied the expression of SirT1 and FoxO3a inhippocampus of CR rats. Furthermore, we applied resveratrol as SirT1 activator toPC12 cells to examine the cell cycle to explore the potential interaction with FoxO3a.Materials and methods:18-month rats were subjected to restricted intake by 60％comparing with that ofrats fed ad libitum (AL) for 6 months. In addition, we examined the expression ofSirT1 and FoxO3a, which mediate important pathways for neuronal growth,senescence and determine the fate of neurons, to explore underlining mechanism ofeffect of CR on aging neurons, using immunohistochemistry and western blot. PC12Cells were analyzed by MTT assay to determine the resveratrol sensitivity at differentconcentration (5, 10, 20, 50μmol/L). SirT1, FoxO3a and p27^KIP1 were measured byRT-PCR after PC 12 cells pretreated with resveratrol at different concentration for 30min. The distribution of cells in the cell cycle was determined by flow cytometryusing propidium iodide-stained nuclei.Results:The level of SirT1 expression was enhanced both in immunohistochemistry(p=0.018) and Western blot (p=0.02). The cytoplasm location of FoxO3a increased inthe hippocampal neurons of the CR rats (p=0.005) showed by immunohistochemistry.In PC12 cell, with increased dose (5-50μmol/L), RSV showed a significant inhibitionin cell proliferation by MTT assay and morphological observation (p＜0.001). Thepercentage of cells in G0/G1 phase tested by flow cytometer increased at the10μmol/L dose of RSV (p=0.023). RT-PCR analysis demonstrated that SirT1 elevatedat the concentration of 10μmol/L RSV (p=0.02). p27^KIP1 elevated with the increaseconcentration of RSV(p＜0.001). The changes of the levels of FoxO3a mRNA didn'treach the statistical significant level (p=0.09).Conclusion:1. Both the unregulated SirT1 and suppressed FoxO3a may participate in postponing the senescence of hippocampal neurons in CR rats.2. SirT1 could be activated by Resveratrol at the concentration of 10μmol/L inPC12 and leads to cell cycle G0/G1 phase arrest.