Shougang Wei, Xiaojuan He, Shaojun Yun, Shuhua Zhang, Zhongxin Xiao

School of Public Health and Family Medicine, Capital Medical University, Beijing  100069, China

Shougang Wei☆, Ph.D., Associate professor, School of Public Health and Family Medicine, Capital Medical University, Beijing  100069, China

Corresponding author: Shougang Wei, School of Public Health and Family Medicine, Capital Medical University, Beijing  100069, China
shangwei@ccmu.edu.cn

Supported by: the Beijing Natural Science Foundation, No. 7052008*

www.crter.cn
www.nrronline.org

doi:10.3969/j.issn.1673-5374.2010.10.012

Abstract
BACKGROUND: Prophylactic dietary restriction (DR), whether lifelong or started in adulthood, retards the aging process and attenuates cognitive decline in rodents. However, whether the an-ti-aging and neuroprotective efficacy of DR initiate late in life or accompany the aging process remains unclear.
OBJECTIVE: The present study sought to: (1) determine if DR could protect against behavioral decline in mice when implemented during the aging process induced by D-galactose and (2) examine neuronal apoptosis in these aged brains and whether DR could block apoptosis.
DESIGN, TIME AND SETTING: The randomized controlled animal study. The experiment was performed at the Experimental Animal Center of Capital Medical University and the Laboratory Center of School of Public Health of Captial Medical University of China from April 2006 to October 2007.
MATERIALS: D-galactose (D-gal) was purchased from Beijing Chemical-Regent Company (Beijing, China). Terminal transferase dUTP nick end labeling (TUNEL) detection kit was obtained from Roche, Germany. Assay kits for antioxidant enzyme activities and malondialdehyde contents were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). Morris water maze (Friends Honesty Life Sciences Co. Ltd., Hong Kong, China) and Flow Cytometry (Coulter, USA) were used in this study.
METHODS: A total of 40 male Institute of Cancer Research (ICR) mice, 3 months old, were equally and randomly divided into D-gal treatment, DR treatment, D-gal + DR treatment and normal control groups, and were then randomly assigned to one of two feeding regimens: ad libitum access to food or DR which received a 70% amount of daily food intake as that by ad libitum fed mice. There were two replicates per feeding regimen and mice were fed for 10 weeks, with or without a daily subcutaneous injection of D-gal at 100 mg/kg.
MAIN OUTCOME MEASURES: Animals’ spatial learning and memory performance were tested in the Morris water maze. Neuronal apoptosis rates were evaluated by Annexin V/flow cytometry  assay and TUNEL assay. Lipid peroxidation levels and antioxidant defense capacity of the brain were measured using testing kits.
RESULTS: DR markedly reduced the prolonged escape latency of D-gal mice in the water maze test (P < 0.01). Annexin V and TUNEL assays showed that the D-gal mice had a significant higher percentage of neuronal apoptosis compared with normal control mice (P < 0.05), and that DR treatment markedly decreased this apoptotic cell death (P < 0.05). DR also reversed the decline of total superoxide dismutase and glutathione peroxidase activities and the increase of malon-dialdehyde levels in the brain of D-gal mice (P < 0.05, respectively).
CONCLUSION: DR reduces the impact of D-gal-induced brain aging in mice and can reverse performance decline and neurobiochemical impairments. These results demonstrate that imple-mentation of DR in conditions of chronic oxidative stress can be neuroprotective, and that senium DR can be beneficial for healthy aging.
Key Words: dietary restriction; brain aging; D-galactose; behavioral performance; neuronal apoptosis; oxidative stress; neural regeneration

INTRODUCTION
  
Dietary restriction (DR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. DR can slow/delay the aging process and increase lifespan[1]. DR may increase the resistance of the nervous system to neurodegenerative disorders and allay many of the adverse effects of aging on the brain[2]. One of the more disconcerting age-related changes is a decline in learning and memory. Lifelong DR attenuates age-related deficits on memory tasks that are hippocampal-dependent in some rodent strains. Furthermore, DR starting in adulthood may also attenuate age-related cognitive decline. However, lifelong or adult DR is of limited practical value since few people will comply with such dramatic eating changes despite the 
tremendous general health benefits. DR intervention initiating at an advanced age (we named it senium DR, relative to lifelong or adult DR) might be more practical, but its anti-aging efficacy is unclear. Therefore, we tested whether a senium DR has any neuroprotective effect.
D-galactose (D-gal) is a naturally occurring substance in the body and is normally metabolized by rodents. However, an overdose of D-gal can be converted to aldose and hydrogen peroxide in animals, leading to the formation of a superoxide anion and oxygen-derived free radicals. D-gal treatment causes oxidative stress in mouse brain and ultimately results in neurodegeneration and cognitive dysfunction[3]. Mice continuously exposed to a high dosage of D-gal show an increase in levels of apoptosis and karyopyknosis in hippocampal neurons and a decline in spatial learning and memory[4-5]. As D-gal induced behavioral and neurochemical changes can mimic many characters of the natural brain aging process, the D-gal-lesioned rodents have been used for brain aging studies[6]. While apoptosis is important in aging, there is no significant age-related decline in hippocampal and neocortical neurons during normal aging[7]. On the other hand, the mechanisms proposed to explain the neuroprotective effects of DR are generally based on studies of longevity and on data from primitive organisms or non-neuronal mammalian tissues. One study in rats actually found a worsening of cognitive function despite increased longevity by DR[8]. In this study, we investigated the effects of senium DR on brain oxidative stress and hippocampal neuronal apoptosis in a D-gal induced aging model to determine whether senium DR can protect or ameliorate the function of the aging brain through its anti-apoptotic as well as antioxidative actions.

MATERIALS AND METHODS

Design
A randomized, controlled, animal study.
Time and setting
The experiment was performed at the Experimental Animal Center of Capital Medical University and the Laboratory Center of School of Public Health of Captial Medical University of China from 2006 April to 2007 October.
Materials
A total of 40 healthy 3 month-old Institute of Cancer Research (ICR) male mice were obtained from and housed at the Experimental Animal Breeding Center of Capital Medical University of China. Specifically, the animal facility was maintained at (21 ± 2) °C, (50 ± 10)% relative humidity, and had a 12 hour light/dark cycle. All mice had free access to water and were weighed weekly. Protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[9].
Reagents and equipment are as follows:

Methods
Animal grouping
After acclimation for 1 week, the animals were equally and randomly divided into D-gal treatment, DR treatment, D-gal + DR treatment, and normal control groups. D-gal treatment and normal control groups were fed a standard diet ad libitum. DR treatment and D-gal + DR treatment groups received a gradually reduced (over 2 weeks) daily food portion until their food intake was reduced to 70% of that received by ad libitum fed mice. D-gal was dissolved in saline to make a 100 mg/mL solution and administered by subcutaneous injection at a dose of 100 mg/kg per day. The non-involved mice (DR treatment and normal control groups) received a daily saline (0.9% NaCl) injection in the same way and amount as D-gal administration. For DR treatment, the diet was vitamin-fortified to ensure a 100% vitamins intake by DR mice as by ad libitum mice. The diet compositions are shown in Table 1.

Morris water maze test
After 10-week intervention, all mice were evaluated behaviorally on the Morris water maze (MWM) test of spatial learning and memory[11]. The MWM system suitably combines evaluation of both spatial memories with locomotor activity. First, animals were trained and tested on the spatial reference memory version of the MWM one by one. For this task, the water tank was divided into four imaginary quadrants of equal sizes (quadrants 1, 2, 3, and 4) with the escape platform located in quadrant 4, 1 cm below the water surface and 28 cm from the edge of the tank. The escape platform remained in the same location throughout the experiment and the mouse was started randomly from quadrants 1, 2, or 3. Swimming behavior was recorded for 120 seconds with a closed circuit video system. For training, five trials were conducted per day for 4 days. In each trial, if the platform was not located within 120 seconds, the mouse was directed to the platform and allowed to stay there for 15 seconds. The following parameters were recorded for the training trials: escape latency to the platform, total distance to platform, and mean swim speed (cm/s).
After the completion of the training trials, the platform was removed from the pool while the visual-spatial environment was left unaltered. A probe test was conducted to assess spatial memory for the location of the escape platform. The animals were allowed to search in the pool for 60 seconds. The following parameters were recorded for the probe test: platform crossing, i.e., the number of crossings through the conceptual location of the platform in each of the quadrants; and target quadrant preference, i.e., the relative distribution of swimming time in each of the quadrants.
Single cell suspension preparation
Mice in each group were anesthetized with chloral hydrate (100 mg/kg, intraperitoneally) and sacrificed following the MWM test. The brain was removed; the cerebral cortex and the hippocampus were separated under a dissecting microscope and placed into a Medicon wetted with 1.0 mL of 1 × phosphate buffered saline (PBS). The Medicon was inserted into the Medimachine and was operated at 100 r/min for 15 seconds. The cell suspension was filtered using a Filcon, a disposable filter device, and washed twice with 1 × PBS. The cells were then counted with a hemocytometer and adjusted to a concentration of 1 × 106 cells/mL.
Annexin V assay 
The apoptosis rate was measured by flow cytometry  using Annexin V-FITC/PI staining methods[12]. In brief, cells were collected by centrifugation, washed once in ice-cold PBS and resuspended in binding buffer at a concentration of 1 × 106 cells/mL, in which 100 μL of cell suspension was added into 5 mL flow cytometry tube. A total of 5 μL of Annexin V-FITC and 10 μL of 20 μg/mL propidium iodide (PI) were added and incubated for 15 minutes in the dark before a further addition of 400 μL of PBS. Quantitative analysis of apoptosis was performed using a flow cytometer with excitation wavelength 488 nm, emission wavelength 515 nm and 610 nm. “AV?PI?” represents the normal cells; “AV+PI?”, the early phase apoptotic cells; “AV+PI+”, the late phase apoptotic cells; and “AV?PI+”, the necrotic cells.
TUNEL assay 
The TUNEL assay[13] was performed for determination of the DNA fragmentation with a Roche TUNEL kit according to its instructions. Briefly, 1 × 106 cells were pelleted by centrifugation, resuspended in 3.7% formaldehyde solution and incubated at room temperature for 10 minutes. Cells were collected by centrifugation, resuspended in 1 × PBS, and incubated for 2 minutes at room temperature. Following another centrifugation, 100 μL of cytonin was added to the cell pellet and incubated for 30 minutes. The cells were washed by TdT labeling buffer, incubated with labeling reaction mix for 1 hour at 37 °C. The reaction was stopped and the cells were incubated with streptavidin-FITC working solution for 10 minutes at room temperature. Cells were centrifuged, resuspended in 500 μL of 1 × PBS, and then submitted to flow cytometry analyses with excitation wavelength 488 nm, emission wavelength 550 nm for FITC.
Assessment of brain antioxidant status and lipid peroxidation
For biochemical studies, the cerebral cortex and hippocampus were homogenized in cold saline. The homogenate (10%, mass fraction) was centrifuged at   4 000 ×g for 10 minutes at 4 °C to collect the supernatant for measurement of superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content using commercial test kits. The activity of total SOD was assayed based on the ability to inhibit oxidation of hydroxylamine. One unit of SOD was defined as the amount of enzyme needed to inhibit 50% of the oxidation of hydroxylamine[14]. GSH-Px activity was measured based on the consumption of glutathione. The reaction of glutathione with dithio-bis-nitrobenzoic acid to produce a yellow benzoic acid anion was catalyzed by GSH-Px, one unit of GSH-Px was defined as the amount that reduced the level of GSH by 1 μmoL in 1 minute[15]. Lipid peroxidation was determined by the formation of MDA, which is a by-product of lipid peroxidation and is widely used as a biomarker of oxidative stress[16], using the thiobarbituric acid method[17]. Protein concentrations were determined by the Bradford method[18]. Values were calculated using optical density, which was monitored in a spectrophotometer (550 nm for SOD, 412 nm for GSH-Px and 532 nm for MDA) and expressed as units U per mg protein for SOD, U per minute per gram protein for GSH-Px, and nmol/mg protein for MDA.
Main outcome measures 
Behavioral performance, neuronal apoptosis rates, lipid peroxidation level, and antioxidant defense capacity of the brain.
Statistical analysis
Results were presented as Mean ± SD. All data were subjected to the Shapiro-Wilk normality test and the homogeneity test of variances. Comparisons between different groups in the behavioral tasks which failed the test of homogeneity were performed using Mann-Whitney U-test. Other data were evaluated using one way analysis of variance, followed by Student’s t-test. P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
No mice showed obvious health problems throughout the whole experimental procedure except one died (without apparent signs of disease) before the completion of the study. Thus, a total of 39 mice were included in the final analysis with 9 – 10 mice in each group.
Effect of senium DR on hippocampal and cortical neuronal apoptosis in aging mice
D-gal mice showed a significantly higher percentage of neuronal apoptosis than D-gal + DR mice and control mice [(23.50 ± 4.35)% in D-gal group, vs. (7.87 ± 1.68)% in D-gal + DR group and (4.26±1.16)% in control group, P < 0.05, respectively], as measured by Annexin V. No significant difference in the percentage of apoptotic neurons was seen between D-gal + DR and control groups or between normal DR [(5.42±0.51)%] and control groups (P > 0.05). The neuronal apoptosis ratio in the cerebral cortex and hippocampus of aging mice was significantly reduced after DR intervention [(13.05±2.22)% in D-gal group, vs. (5.64 ± 1.09)% in D-gal + DR group, P < 0.05], but the DR group [(4.32 ± 0.91)%], control group [(3.68 ± 0.69)%] and D-gal + DR group were similar in the TUNEL assay (P > 0.05) (Figure 1). 

Effect of senium DR on the oxidative/antioxidative status in aging mice
Malonaldehide levels in the cerebral cortex and hippocampus homogenate of D-gal mice were significantly greater than controls (P < 0.05). The increase in lipid peroxidation indicated elevated oxidative stress in the brain of D-gal mice. DR could attenuate D-gal induced increase in the MDA level (P < 0.05), but there was no statistically significant difference between DR and the normal control groups (Table 2).
In D-gal mice, total SOD and GSH-Px activities declined significantly compared with the controls (P < 0.05, respectively). In contrast, no significant decrease in activities of these antioxidant enzymes was observed in D-gal +DR mice, indicating that DR preserved antioxidant activities in mice exposed to D-gal.

Body weight changes caused by DR and D-gal
All ad libitum (control and D-gal) mice gained weight over time, whereas those on the DR regimen maintained their initial weight throughout the feeding period. At the end of the experiment, the mean weights of DR-treated (both DR and D-gal + DR) mice were lower compared to control mice (P < 0.05). D-gal and control groups showed similar weight increases (P > 0.05; Figure 2).

Effect of senium DR on behavioral performance
The D-gal group showed an increased latency to the platform compared with controls (P < 0.01), indicating impaired spatial learning and memory. The D-gal + DR mice had shorter escape latency than DR mice        (P < 0.01). This was consistent with the result of total travel distance to the platform during training days (data not shown). There was no significant difference in escape latency between normal control and D-gal + DR groups, nor was between normal control and DR animals (Figure 3).

The D-gal group had significantly slower swimming speed compared with the normal control and the D-gal + DR groups (P < 0.01). The differences in locomotor ability of mice contributed to the latency differences during the training days. Therefore, DR led to the shortened latency to the platform by, at least partly, improving the D-gal-impaired locomotor activity.
In the probe trial, both the D-gal-treated and the D-gal + DR mice made fewer platform crossings than the control group (P < 0.05), and DR and control mice had similar numbers of platform crossings (Figure 4A). Furthermore, both the D-gal and the D-gal + DR mice had a lower spatial preference for the target quadrant than the control group (P < 0.05). Meanwhile, no significant difference on quadrant preference was determined between DR and control mice (Figure 4B).

DISCUSSION

The present results demonstrate for the first time that senium DR during the brain aging process can protect against behavioral performance decline in mice, probably by, at least in part, attenuating oxidative stress induced neuronal apoptosis and preserving the antioxidant defense capacity of the brain. Oxidative stress, which results from an imbalance in the oxidant/antioxidant system favoring the former, is an essential source of cellular injury in the brain. In this study, we used the D-gal-lesioned mouse as a brain aging model for studying the anti-aging effect of DR. D-gal increased malonaldehide levels, an indicator of oxidative stress in the mouse brain, as seen in a previous study[19]. D-gal also decreased SOD and GSH-Px activities, as shown previously[20]. Decreased SOD activity may be due to the inactivation of this enzyme by H2O2[21], which is a product of D-gal in the presence of galactose oxidase, and the decrease in GSH-Px activity may be attributed to the accumulation of the superoxide anion, which inactivates GSH-Px by reacting with selenium at the active site of this enzyme[22]. DR blocks D-gal toxicity by increasing SOD and GSH-Px activities and by decreasing malonaldehide production. DR may protect antioxidant enzymes from being inactivated by overdose of D-gal or may increase the gene expression of antioxidant enzymes but not affect activity.
The MWM, typically consisting of spatial learning acquisition training and spatial accuracy memory in a probe trial, is one of the most reliable laboratory tools in behavioral measures of cognitive function. The present study focused specifically on latency to the platform for the more detailed evaluation of performance because many of the studies that assessed chronic adult DR effects in spatial learning processes relied primarily on latency measures. Here, DR treatment accompanying the injection of D-gal could reverse the spatial learning impairment as indexed by escape latency in acquisition training. Clearly, this result suggested a therapeutic action of DR on spatial learning impairment induced by D-gal. In contrast, results of the probe test revealed little, if any, DR effects on cognitive function, as indexed by measures of preference and accuracy, indicating that DR did not affect D-gal-induced declines in accuracy of spatial navigation. The inconsistency of results in acquisition training and probe trial suggests that the beneficial effect of DR on escape response latency is an index of the preservation of motor, but not cognitive, function. Another explanation for DR effects on acquisition performance in the absence of exhibiting superior probe performance is that, as analyzed by Fitting et al[23], DR might be effective in enhancing either learning or working memory process (acquisition training), without affecting spatial or long term memory (probe test). Here, DR reversed declines in behavioral performance, reflecting amelioration of brain function.
D-gal incudes apoptosis in cortical and hippocampal neurons. Neuronal loss from the cortex and hippocampus by apoptosis may mediate D-gal-induced impediments in motor and cognitive functions, since these are the brain regions related to motor and cognitive performance. Moreover, DR treatment rescues cerebral neuron loss in these aging models, which may contribute to the protective effect of DR on D-gal-induced behavioral retrogression. DR could reduce the production of reactive oxygen species or mediate a mild stress response in neurons, presumably because of a decreased energy availability to the neurons, leading to increased production of neuroprotective proteins and better abilities to cope with oxidative stress and apoptotic death[24]. DR may therefore affect the oxidant/antioxidant balance as well as the anti-apoptotic potency of the brain, along with the improving cognitive and motor performance. Putting a DR regimen into practice under increased oxidative stress, which is a characteristic of aging, would be a feasible neuroprotective measure.
Experiments in species as diverse as yeast, worms, flies rodents and primates have revealed that lifelong or adult DR prolongs survival and counteracts the aging process[25-27]. In regard to senium DR, few papers have focused on its role in extending lifespan and the outcomes are inconsistent[28]. Implementation of DR accompanying the oxidative stress-induced aging process in mice can protect the animals from behavioral and neurochemical disorders. That is to say, senium DR would be good for the protection of brain function. Aging is plastic[29], anti-aging interventions do not have to be lifelong. This notion was supported by research[30] showing that when fruit flies fed a restricted diet were changed to a full diet, mortality increased to the level suffered by fully fed flies. Conversely, when the diet of fully fed flies was restricted, mortality decreased to the level enjoyed by flies that have experienced a lifelong restricted diet. Another recent report, asserted therapeutic implementation of DR in rat after spinal cord injury was neuroprotective[31], further strengthen the opinion that it is never too late to initiate a DR regimen for its multiple benefits. Just as our data indicated, senium DR was neuroprotective and could switch the brain aging process to a slower, healthier trajectory.

REFERENCES

[1] Merry BJ. Dietary restriction in rodents-delayed or retarded ageing? Mech Ageing Dev. 2005;12(9):951-959.
[2] Ingram DK, Young J, Mattison JA. Calorie restriction in nonhuman primates: assessing effects on brain and behavioral aging. Neuroscience. 2007;145(4):1359-1364.
[3] Lu J, Zheng YL, Luo L, et al. Quercetin reverses D-galactose- induced neurotoxicity in mouse brain. Behav Brain Res. 2006;171(2):251-260.
[4] Wu DM, Lu J, Zheng YL, et al. Purple sweet potato color repairs D-galactose-induced spatial learning and memory impairment by regulating the expression of synaptic proteins. Neurobiol Learning Mem. 2008;90(1):19-27.
[5] Lu J, Zheng YL, Wu DM. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by D-galactose. Biochem Pharmacol. 2007;74(7): 1078-1090.
[6] Hua XD, Lei M, Zhang YJ, et al. Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimer's disease. Life Sci. 2007;80(20):1897-1905.
[7] Calhoun ME, Kurth D, Phinney AL, et al. Hippocampal neuron and synaptophysin positive bouton number in aging C57BL/6 mice. Neurobiol Aging. 1998;19(6):599-606.
[8] Yanai S, Okaichi Y, Okaichi H. Long-term dietary restriction causes negative effects on cognitive functions in rats. Neurobiol Aging. 2004;25(3):325-332.
[9] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[10] American Institute of Nutrition. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of AIN-76A rodent diet. J Nutr. 1993;123(11):1939-1951.
[11] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984;11(1):47-60.
[12] Van HWL, Robert OS, Dumont E, et al. Markers of apoptosis in cardiovascular tissues: Focus on Annexin V. Cardiovasc Res. 2000;45(3):549-559.
[13] Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119(3):493-501.
[14] McCord JM, Fridowich I. An enzymic function for erythrocuprein. J Biol Chem. 1969;244(22):6049-6055.
[15] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70(1):158-169.
[16] Cini M, Fariello RG, Bianchetti A, et al. Studies in lipid peroxidation in the rat brain. Neurochem Res. 1994;19(3):283-288.
[17] Uchiyama M, Mihara M. Determination of malondialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem. 1978;86(1):271-278.
[18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
[19] Xiuli Z, Bo J, Zhibo L, et al. Catalpol ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by D-galactose. Pharmacol Biochem Behav. 2007;88(1): 64-72.
[20] Fan SH, Zhang IF, Zheng YL, et al. Troxerutin protects the mouse kidney from D-galactose-caused injury through anti-inflammation and anti-oxidation. Int Immunopharmacol. 2009;9(1):91-96.
[21] Hodgson EK, Fridovich I. The interaction of bovine superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochem. 1975;14(24):5294-5299.
[22] Prabhu HR. In vivo inhibition of selenium dependent glutathione peroxidase and superoxide dismutase in rats by diethyldithiocarbamate. Ind J Exp Biol. 2002;40(3):258-261.
[23] Fitting S, Booze RM, Gilbert CA, et al. Effects of chronic adult dietary restriction of spatial learning in the aged F344 Χ BN hybrid F1 rat. Physiol Behav. 2008;93(3):560-569.
[24] Sharma S, Kaur G. Neuroprotective potential of dietary restriction against kainate-induced excitotoxicity in adult male Wistar rats. Brain Res Bull. 2005;67(6):482-489.
[25] Masoro EJ. Caloric restriction-induced life extension of rats and mice: A critique of proposed mechanisms. Biochim Biophys Acta. 2009;1790(10):1040-1048.
[26] Oita RC, Mazzatti DJ, Lim FL, et al.Whole-genome microarray analysis identifies up-regulation of Nr4a nuclear receptors in muscle and liver from diet-restricted rats. Mech Ageing Dev. 2009;130(4):240-247.
[27] Colman RJ, Anderson RA, Johnson SC, et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science. 2009;325(5937):201-204.
[28] Yen K, Mobbs CV. Chemosensory and caloric mechanisms influence distinct components of mortality rate. Exp Gerontol. 2008;43(12):1058-1060.
[29] Vaupel JW, Carey JR, Christensen K. Aging: It’s never too late. Science. 2003;301(5640):1679-1680.
[30] Mair W, Goymer P, Pletcher SD, et al. Demograph of dietary restriction and death in Droophila. Science. 2003;301(5640): 1731-1733.
[31] Plunet WT, Femke SF, Lam CK, et al. Dietary restriction started after spinal cord injury improves functional recovery. Exp Neurol. 2008;213(1):28-35.
(Edited by Yu XP/Qiu Y/Song LP)