Rhesus monkey ageing and CR study

For rhesus monkeys in captivity, median survival is ~26 years of age, 10% survival is ~35 years of age and maximum survival is ~40 years of age8. The rhesus monkey CR and ageing study was initiated in 1989 at the Wisconsin National Primate Research Center (WNPRC)16. Animals were recruited in two phases, the first containing 30 males and the second phase, begun in 1994, in which 30 females and an additional 16 males were added. Animals in all groups were adults when introduced into the study (7–14 years old). The classic rodent CR experiment, in which lifespan may be extended by as much as 40%17, involves animals being placed on the diet at an early age just after weaning. Despite initial uncertainties as to whether adult-onset CR would also prove beneficial in terms of lifespan, careful studies have shown that with gradual reduction in food intake adult-onset of CR is almost as effective as early-onset in extending lifespan in rodents18. The adult-onset CR model was chosen for the rhesus monkey study on the basis that any potential ageing intervention in human studies would be initiated in adult populations. Within each of the three groups, animals were randomized to control or CR diets taking into consideration baseline food intake, body weight and age. Individualized food allotments were calculated based on daily food intake data that were collected for each animal over a 3–6-month period before initiation of the dietary intervention. For each animal in the CR groups, the individual intake determined at baseline was reduced by 10% per month over a 3-month period to reach the desired 30% restriction.

CR reduces age-related and all-cause mortality

To assess the impact of CR on age-related and all-cause survival, the cause of death for each monkey was determined on necropsy by a board-certified pathologist who was blinded to the animal’s diet group. Deaths due to acute conditions clinically unrelated to ageing were excluded from the age-related mortality analysis but were retained for the evaluation of all-cause mortality. Of the original 76 animals, 63% (24/38) of the control animals died of age-related causes compared with only 26% (10/38) of the CR group. Considering only age-related deaths and excluding animals with non-age-related deaths (NARDs), a statistically significant effect of CR in increasing survival was observed (Cox regression P=0.007; Fig. 1a) with a hazard ratio (HR) of 2.89 (95% confidence interval (CI): 1.34–6.25) indicating that at any point in time the control animals had 2.9 times the rate of death from an age-related cause when compared with animals under CR. Conducting this same analysis including NARDs but treating them as censored values also revealed a statistically significant effect of CR in increasing survival (Cox regression P=0.001) with a HR of 3.63 (95% CI: 1.69–7.81).

Figure 1: Mortality curves. (a) Age-related mortality. Animals that died from non-age-related causes are excluded. The Cox regression detected a statistically significant effect of CR in increasing survival (P=0.007) with a HR of 2.89 (95% CI: 1.34–6.25). (b) All-cause mortality. These curves depict data for all animals on the study not censored for cause of death. The Cox regression detected a statistically significant effect of CR (P=0.037; Fig. 1b) with an estimated HR of 1.78 (95% CI: 1.04–3.04). Full size image

Survival analysis showed that effect of CR on all-cause mortality is also statistically significant (Cox regression P=0.037; Fig. 1b) with an estimated HR of 1.78 (95% CI: 1.04–3.04) indicating that at any point in time the control animals had 1.8 times the rate of death from any cause when compared with animals under CR. In all, 8 controls and 16 animals subjected to CR died of non-age-related causes, which included complications of anaesthesia, gastric bloat, endometriosis and injury. Although twice as many CR animals died from a NARD, there were twice as many CR animals alive at this stage of the study. For animals younger than 20 years of age the incidence of NARD was 11% for control animals and 13% for CR. A competing risk model that treated age-related and non-age-related deaths as mutually exclusive events indicated that CR animals were not significantly more likely to die from a non-age-related cause (competing risk model P=0.373). Applying the same analysis to age-related deaths confirmed the previous result of the censored analysis, in that the effect of CR on age-related mortality was statistically significant (competing risk model P=0.001) with HR of 3.62 (95% CI: 1.69–7.82). Differences in survival of the CR and control animals are shown in Table 1. Estimates of lifespan were determined using the Kaplan–Meier statistical model and parametric survival analysis assuming a Weibull distribution, with both indicating improved survival for monkeys on CR. Importantly, the median survival of control animals (~26 years) is comparable to that of animals at WNPRC not participating in this study, demonstrating that CR has increased survival relative to normal rhesus monkey ageing.

Table 1 Estimates of lifespan. Full size table

Comparison of control monkeys from UW and NIA CR studies

Recently, the results of the NIA rhesus monkey young-onset and old-age-onset CR study were published12, and although modest benefits in overall measures of health and function were observed in CR animals compared with controls, a significant difference in survival was not detected. To try to understand the differences in outcome, we focused on the differences in the study design. The implementation of the CR diet is a major point of divergence between the UW and NIA studies. The primary goals of the UW study were to investigate rhesus monkeys as a model for human ageing and to determine the impact of CR on ageing. The UW control animals were ad libitum fed to mirror human feeding habits. The NIA study is closer to the classic CR study design used in rodent studies; the study included early-onset cohorts and NIA control animals were fed according to regulated portioning rather than ad libitum. We compared body weight data for control animals from the NIA study that had been previously published19,20 with age- and gender-matched control animal data from the UW study (Fig. 2a). NIA female monkeys weighed significantly less than UW controls both at 12–18 and 21–25 years of age (Welch’s t-test P=0.014, P=0.049). Male monkeys of 10–12 years age from the NIA study also weighed less than those from the UW study (Welch’s t-test P=0.003). The body weight difference between an older age group of males showed the same trend but was not statistically significant (24–33 years, Welch’s t-test P=0.169); however, the reduced size of both cohorts (n=7 NIA and n=4 UW) and the large variance among individuals at advanced age limits the possibility that statistical significance could be observed.

Figure 2: Body weight comparisons. (a) Body weights for male and female control animals from UW and NIA studies. Males, 10–12 years: NIA=6, UW=15; 24–33 years: NIA=7, UW=4. Females, 12–18 years: NIA=20, UW=15; 21–25 years: NIA=5, UW=8. Data are shown as average±s.e.m. *P<0.05 (Welch’s t-test). (b) Comparison of body weights for control animals on UW and NIA rhesus monkey ageing and CR studies with national averages for age- and gender-matched animals. Data shown in (a) are presented as per cent deviation from averages for males aged 10–12 years (175 animals, 2,790 data points), males aged 24–33 years (114 animals, 11,805 data points), females aged 12–18 years (334 individuals, 18,626 data points) and females aged 21–25 years (255 animals, 15,666 data points). Full size image

Comparison of CR studies with national monkey ageing data

To place these data in a broader context, we next determined how the control animals from both studies compared with the national average for body weight for male and female rhesus monkeys within these age groups. The internet Primate Ageing Database (iPAD; http://ipad.primate.wisc.edu/) is a joint initiative of the NIA (intramural and extramural programs), NIH Office of Research Infrastructure Programs (ORIP) and the National Primate Research Center at the University of Wisconsin–Madison. The iPAD hosts clinical and biometric data from healthy, non-experimental, captive non-human primates housed in research facilities across the USA. Body weight averages of age- and gender-matched animals were calculated on the basis of iPAD data from 878 individuals using 48,887 data points (Fig. 2b). Although UW and NIA intramural studies both exclusively housed animals individually, the iPAD does not distinguish between group and individual housing so that data from iPAD included in comparison with UW and NIA intramural studies were not limited to individually housed animals. For each gender and age group, the UW control animals consistently weighed more than average. In contrast, the body weights of the NIA control animals were consistently lower than average for both genders and at both age groups considered. One explanation for the lack of difference in survival among animals from the NIA study is that the control animals were in fact restricted and the advantages of mild restriction on survival in the control animals were not improved by further restriction in the CR group. Data from the NIA old-age-onset male cohort is consistent with improved survival in both control and CR groups. Of the 20 male monkeys initiated into the study in old age, 5 lived beyond 40 years of age, 1 from the control group and 4 from the CR group. In over three decades of maintaining an ageing colony of >100 animals at the WNPRC, only one monkey with a known birth date reached 40 years of age. Furthermore, of the 3264 rhesus monkeys (175,456 data points) in the iPAD only two 40-year-old animals have been documented. These data indicate that the five 40-year-old monkeys from the NIA intramural study are exceptionally long-lived.