Frailty Quantified by the “Valencia Score” as a Potential Predictor of Lifespan in Mice

Frailty Quantified by the “Valencia Score” as a Potential Predictor of Lifespan in Mice Abstract The development of frailty scores suitable for mice and which resemble those used in the clinical scenario is of great importance to understand human frailty. The aim of the study was to determine an individual frailty score for each mouse at different ages and analyze the association between the frailty score and its lifespan. For this purpose, the “Valencia Score” for frailty was used. Thus, a longitudinal study in mice was performed analyzing weight loss, running time and speed, grip strength and motor coordination at the late-adult, mature and old ages (40, 56 and 80 weeks old, respectively). These parameters are equivalent to unintentional weight loss, poor endurance, slowness, weakness, and low activity level, respectively, in humans. A cut-off point was used to identify frail mice for each criterion. All the measurements were also performed on chronologically adult prematurely aging mice. The results show that by using the “Valencia Score” for frailty a prematurely aged phenotype can be identified even during the adulthood of animals. This opens up the possibility of carrying out preventive long-term interventions. Moreover, the individual frailty score of a given mouse at the late-adult, mature and old ages is shown to be a relevant predictor of its lifespan. Rate of aging, Longevity, Experimental models In the last decades, the aim of health care has switched from trying to live longer, to experiencing healthy aging. This is due to the current higher life expectancy being accompanied by an increase in disability rates and consequently, the lack of independence, autonomy, and well-being (1). Disability is often preceded by a state characterized by a diminished ability to respond to different stressors, which has been termed as frailty. In this regard, the current goal of improving healthy life expectancy is to act before disability arises by preventing or delaying the onset of frailty (2). Thus, frailty is a clinical geriatric syndrome defined by a diminished ability to restore homeostasis after any physical or mental damage, especially due to the absence of regulation of several physiological systems. The consequence of this being that when one individual faces minor stress situations, it results in outcomes like hospitalization, disability, and finally, death (3–7). Another important characteristic of frailty is that it is reversible, that is, it can be treated and even prevented. This is the reason why it has become necessary to determine which subjects are “frail” in the clinical scenario. In humans, there is a wide number of different scales to quantify frailty with different degrees of difficulty and clinical applicability (8). The two most commonly used ones in the clinical scenario are the phenotype frailty score and the frailty index (FI) based on deficit accumulation. The phenotype frailty score, developed by Fried and colleagues (3), takes into account five criteria: unintentional weight loss, slow walking speed, self-reported exhaustion, weakness and low physical activity. Individuals which meet three or more of these five criteria are classified as frail, individuals that meet one or two are classified as prefrail and those individuals with none are categorized as robust (3). In contrast, the FI, based on deficit accumulation, counts the number of potential health deficits of an individual and divides them by the total number of items measured (9). Another difference is that in the deficit accumulation approach, the reference values used are those of an adult, whereas in the phenotype frailty score, the reference values of the criteria are those corresponding to the age group studied. The development of frailty scores suitable for mice and which resemble those that are used in the clinical scenario has become an essential challenge in basic gerontological research (10), given that these would be useful tools to assess the effect of a given intervention in mice before translating it to humans. Thus, some frailty scores have been developed for the quantification of frailty in mice. Most of them have followed the deficit accumulation approach (9), by counting the presence or absence of a different set of deficits in mice (11–15). Following Fried’s phenotype frailty score (3), a frailty score has also been developed for the measurement of frailty in mice. It was first proposed by Liu and colleagues (16) and further developed by Gómez-Cabrera and colleagues (17). This last frailty score, which has been named the “Valencia Score” (17), adapts the same criteria to mice that are taken into account in humans (unintentional weight loss, endurance, slowness, weakness, and low activity level). Thus, weight loss, running time and speed, grip strength, and motor coordination were measured in mice. As it takes into account criteria that are closely related to the clinically accepted frailty score and it is simple to use; it may facilitate frailty research in animal models. Moreover, the results would have the potential to translate to clinical settings. Although some of the above-mentioned frailty indices, based on the deficit accumulation approach (14,15), have been shown to be predictive of mortality in mice, it is still not known if the “Valencia Score” would also be able to make this prediction. Most of the research performed on frailty has been focused on elderly people or mice. However, it is known that the age-related deterioration of the physiological systems does not occur at the same rate in all the individuals of a population with the same chronological age (18). Thus, subjects with the same chronological age can show large differences regarding health status and functional capacity and these interindividual differences can already be quantified at the adult age. This has been recently shown both in humans (19,20) and in mice (12,14,15). Even though some interventions initiated at the old age have been shown to be beneficial in decreasing the frailty status (13,17), the identification of frail individuals at the adult age would enable earlier interventions, which potentially could show more beneficial effects. However, in the “Valencia Score” for frailty, only 17 months old mice and older were studied (17) and it is still not known if it can be used to detect differences in the frailty status within younger mice. In agreement with the heterogeneity of the aging process, previous studies from our group have proposed a natural murine model of premature aging based on an inappropriate reactivity to stress. Thus, when mice are submitted to a simple T-maze test, those that need more time to explore it are classified as prematurely aging mice (PAM), whereas those that show an exceptional response to the maze, needing less time to explore it, are classified as exceptional non-PAM (E-NPAM). In addition, those mice that show an intermediate behavior, which constitute around 80 per cent of the population, are classified as regular non-PAM (R-NPAM). So far, it has been demonstrated that these PAM at the adult age show premature immunosenescence (21,22), an altered neurochemistry (23), higher anxiety-like behaviour (21,24), skeletal alterations (25), higher oxidative stress levels (21,26), and a shorter lifespan (27,28), compared with their counterpart E-NPAM of the same sex and chronological age (reviewed in Refs. 29 and 30). Hence, the first objective of the present work was to validate the “Valencia Score” in another sex and strain of mice (outbred female ICR-CD1) not previously studied, using a longitudinal study. Thus, a group-frailty score for each age (late-adult, mature, and old) was calculated. A group-frailty score was also calculated for E-NPAM and PAM at the late-adult age. The second objective was to determine an individual frailty score for each mouse at the late-adult, mature, and old ages (using as cut-off values the corresponding performance at each age) throughout a longitudinal study as well as to analyze the association between the frailty score of each mouse at a given age and its respective lifespan. For the longitudinal study, only R-NPAM were used. Materials and Methods Experimental Animals Female ICR/CD1 ex-reproductive mice (Mus musculus) of 32 ± 4 weeks were purchased from Janvier Labs (Germany) and placed in the Animal Facility at the Faculty of Biology (Universidad Complutense de Madrid, UCM). Mice were housed at 4–5 per cage. The average temperature in the Animal Facility was 22 ± 2°C, relative humidity was 60 per cent, and a 12/12 hour reversed light/dark cycle (lights on at 20:00h) was maintained to avoid circadian interferences. Mice were checked daily. Water and standard pellets (Panlab, Spain) were available ad libitum. Classification of mice into E-NPAM, R-NPAM, and PAM using a T-maze test After 1 week of acclimatization following the arrival of late-adult female ICR/CD1 mice (33 ± 4 weeks), the classification of these animals into PAM and NPAM was carried out. Previous experience in our laboratory has shown that 15 ± 5% of mice purchased behave as PAM and another 15 ± 5% behave as E-NPAM. Thus, 80 mice were submitted to the T-maze test in order to obtain enough mice from each type. The T-shaped maze is composed of three wooden arms (each 10 cm wide, 25 cm long, and 10 cm high) covered with black methacrylate. The floor consists of cylindrical aluminum rods that are 3 mm thick and arranged perpendicularly on the side walls. The test was carried out by holding the mouse by its tail and positioning it inside the base of the “T” with its head facing the end wall. Then, the time that each mouse took to cross the intersection of the “T” with both hind legs was measured. This test was performed once per week for a month, to distinguish PAM (which needed more than 10 seconds to cross the intersection at each test the four times) from the NPAM, as described previously (27,28). Depending on the behavior of NPAM, they were divided into E-NPAM (that required less than 10 seconds to cross the intersection at each test the four times) and R-NPAM (those that show an intermediate behavior spending less than 10 seconds sometimes and others more than 10 seconds). This test was always carried out under red light and between 09:00 and 11:00 hours in order to avoid circadian variations. Out of the 80 mice, 60 behaved as R-NPAM (75%), 10 as E-NPAM (12.5%), and 10 as PAM (12.5%). Not all mice were used for the study, only the following groups. One group of R-NPAM (n = 10) was used for the determination of the reference values for the “Valencia Score” at three different ages (40, 56, and 80 weeks, respectively). Another group of animals (n = 20), also R-NPAM, was used for the longitudinal study. The “Valencia Score” was carried out at the ages previously mentioned. Maximum lifespan in R-NPAM was 112 weeks with an average lifespan of 76 weeks. The 40 week old mice can be considered as late-adult mice, the 56 week old mice as mature mice, and the 80 week old animals as old mice. The “Valencia Score” test was also performed in 10 PAM and 10 E-NPAM at the late-adult age (40 ± 4 weeks) only. All the procedures were approved by the Experimental Animal Committee of UCM (Spain) and were in consonance with the European Community Council Directives 2010/63/EU of 22 September 2010 guidelines. Body Weight The body weights of the mice were recorded individually 1 month prior to and at the moment of carrying out the “Valencia Score” for frailty. When a given mouse lost more than 5 per cent of its weight compared with the previous month, it was considered frail for this criterion. This cut-off was selected in order to resemble the Fried’s phenotype frailty score for humans, which considers the loss of 5 per cent of body weight in the previous year as a frailty criterion (3). Motor Coordination Test The tight-rope test described by Miquel (31) and widely used by others (32) was used with the following modifications. Mice were placed in the middle of a bar of circular section (60cm long and 1.5cm diameter) and the test was considered successful if the mouse was able to reach either the end of the bar, or if it did not fall, during a period of 60 seconds in at least one out of the five consecutive trials. Incremental Treadmill Test Mice were submitted to an intensity treadmill test (Model LE8706, Panlab). The protocol of Davidson and coworkers (33) was used with the following modifications. Mice were placed on the treadmill at an initial speed of 6 m × minutes−1 for 6 minutes (warm-up period). After it, the treadmill band speed was increased by 2 m × minutes−1 every 2 minutes until the animals were exhausted. Exercise motivation was administered by the presence of an electric shock grid at the base of the treadmill. Exhaustion was defined as the refusal to run after three consecutive tail shocks. The maximum running time and running speed were recorded as measurements of their endurance and slowness, respectively. Grip Strength Test The Grip Strength Meter (Panlab. Harvard Apparatus) was used to measure the maximum force displayed by a mouse. Briefly, the grip strength meter was positioned horizontally and mice were held by the tail and lowered toward the apparatus. Animals were allowed to grasp the metal bar with their forelimbs and then they were pulled backwards in the horizontal plane (34). Maximum peak force of each mouse was automatically registered in grams-force. The test was performed five consecutive times. Since the weight influences the force of an animal, the net grip strength was adjusted by dividing the registered force by the weight of the animal. Data Analysis Analysis was performed with SPSS 21.0 (SPSS, Chicago, USA) software. Normality of the samples was checked by the Kolmogorov–Smirnov test and homogeneity of the variances was checked by the Levene test. Age-related differences in running time, running speed, and grip strength were studied using a one-way analysis of variance followed by post hoc analysis. The Tukey test was used for post hoc comparisons when variances were homogeneous, whereas Games–Howell analysis was used when variances were not homogeneous. Age-related differences regarding the tight-rope test and weight loss, as well as in the prevalence of frailty, were analyzed using Pearson’s chi-squared test. Differences in lifespan were investigated using the Kaplan–Meier test, with a minimum significance level (log rank, Mantel-Cox) set at p < .05. Two-sided p < .05 was considered the minimum level of significance. Results Group-Frailty, Quantified by the “Valencia Score”, Is Higher in PAM and Correlates With Lifespan The results show that both mature and old R-NPAM experience a decrease in the running time (endurance) as well as in the running speed (slowness) compared with when they are late-adults (p < .01 for mature mice; p < .001 for old mice). Old mice also experience a decrease in both components compared with when they are mature (p < .05 in running time; p < .01 in running speed) (Figure 1A and C). Regarding the premature aging model, chronologically adult PAM show lower running time and speed than E-NPAM (p < .05), whereas no statistically significant differences are found between PAM and late-adult R-NPAM (Figure 1B and D). Strikingly, E-NPAM show a higher running time than the late-adult R-NPAM (p < .05). Figure 1. View largeDownload slide Running time (A), running speed (C), grip strength (E), and net grip strength (G) values in R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The values corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B), (D), (F), and (H), respectively. These values were only compared with those of R-NPAM at the adult age. Statistical differences were tested using one-way ANOVA. a: p < .05; aa: p < .01; aaa: p < .001 with respect to the values in late-adult mice. b: p < .05; bb: p < .01 with respect to the values in mature individuals. c: p < .05; cc: p < .01 with respect to the values in E-NPAM. Figure 1. View largeDownload slide Running time (A), running speed (C), grip strength (E), and net grip strength (G) values in R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The values corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B), (D), (F), and (H), respectively. These values were only compared with those of R-NPAM at the adult age. Statistical differences were tested using one-way ANOVA. a: p < .05; aa: p < .01; aaa: p < .001 with respect to the values in late-adult mice. b: p < .05; bb: p < .01 with respect to the values in mature individuals. c: p < .05; cc: p < .01 with respect to the values in E-NPAM. Weakness is another key component of the diagnosis of clinical frailty (3). Thus, weakness of mice was investigated by measuring grip strength. As shown in Figure 1E, there are no age-related changes regarding grip strength in R-NPAM, whereas PAM show lower values than E-NPAM and late-adult R-NPAM (p < .05) (Figure 1F). There are many studies, all of them carried out in humans, which have found a positive correlation between grip strength and BMI or weight (35–37). Thus, in order to avoid differences in grip strength due to differences in body weight, a net grip strength variable was calculated by dividing the peak force registered for each animal by its weight. The results (Figure 1G) show that old R-NPAM experience a decrease regarding net grip strength compared with when they are late-adult (p < .05). In regard to the premature aging model, chronologically adult PAM show lower net grip strength than E-NPAM and R-NPAM (p < .01, p < .05, respectively) (Figure 1H). Strikingly, E-NPAM show higher net grip strength than late-adult R-NPAM (p < .001). The tight-rope test is a broadly used and extensively validated behavioral marker of aging (32,39). The modified protocol used in the present study was considered a good marker of motor coordination, which was quantified as the percentage of mice that succeeded in performing the test (17). In addition, unintentional weight loss was quantified as the percentage of mice that succeeded in not losing more than 5 per cent of their body weight compared with the previous month. The results (Figure 2A) show that old R-NPAM experience a decrease in the percentage of success in performing the tight-rope test in comparison to when they are late-adult (p < .01) and mature (p < .05). PAM have a lower percentage of success than E-NPAM, although nonstatistically significant differences are found (Figure 2B). Regarding weight loss, a tendency towards an age-related decrease in the ability to maintain body weight is observed, although no statistically significant differences are found at the different times of study in R-NPAM as well as in PAM (Figure 2C and D). Figure 2. View largeDownload slide Percentage of success in the tight-rope test (A) and unintentional weight loss (C) of R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The results corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B) and (D), respectively. Data regarding weight loss are expressed as percentage of mice that did not lose more than 5 per cent of their weight compared with the previous month. Statistical differences were tested using Pearson’s chi-squared test. aa: p < .01 with respect to the values in late-adult mice. b: p < .05 with respect to the values in mature individuals. Figure 2. View largeDownload slide Percentage of success in the tight-rope test (A) and unintentional weight loss (C) of R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The results corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B) and (D), respectively. Data regarding weight loss are expressed as percentage of mice that did not lose more than 5 per cent of their weight compared with the previous month. Statistical differences were tested using Pearson’s chi-squared test. aa: p < .01 with respect to the values in late-adult mice. b: p < .05 with respect to the values in mature individuals. Based on the performance of mice at each age studied, reference values were obtained for each age by selecting the value of the 20th percentile for the variables running speed, running time, and net grip strength (Figure 3A). The variable net grip strength (grip strength/weight) was used instead of grip strength, given that the first showed a more marked age-related decrease (Figure 1E and G). Therefore, each mouse that ranked below the 20th percentile for any of these criteria was considered as a failure for that criterion. Regarding motor coordination and weight loss components, each mouse that failed to complete the tightrope test or lost more than 5 per cent of its body weight compared to the previous month, respectively, was considered as a failure for that criterion. Figure 3. View largeDownload slide (A) Reference values applied for each age investigated. (B) Kaplan–Meier cumulative survival curves of E-NPAM (n = 10), R-NPAM (n = 20), and PAM (n = 10). (C) Group-frailty score for adult, mature, and old R-NPAM obtained using, respectively, adult, mature, and old cut-off values as references. (D) Group-frailty score for chronologically adult E-NPAM and PAM obtained using adult cut-off values as references. Statistical differences between lifespans were tested using the Kaplan–Meier log‐rank test. **: p < .01; ***: p < .001. Figure 3. View largeDownload slide (A) Reference values applied for each age investigated. (B) Kaplan–Meier cumulative survival curves of E-NPAM (n = 10), R-NPAM (n = 20), and PAM (n = 10). (C) Group-frailty score for adult, mature, and old R-NPAM obtained using, respectively, adult, mature, and old cut-off values as references. (D) Group-frailty score for chronologically adult E-NPAM and PAM obtained using adult cut-off values as references. Statistical differences between lifespans were tested using the Kaplan–Meier log‐rank test. **: p < .01; ***: p < .001. For the calculation of the frailty score for each age group (late-adult, mature, and old R-NPAM), the corresponding age-matching reference values were applied. Each group-frailty score, expressed as a percentage, was obtained by dividing the total number of tests failed by the mice of each age, by the total number of tests performed by these mice. As can be seen in Figure 3C, the frailty score for late-adult R-NPAM is 15, for mature R-NPAM is 21, and for old R-NPAM is 25. These frailty scores cannot be compared among them, and they just represent an average frailty score for mice at these ages. In addition, following the same procedure described above, group-frailty was also calculated for adult PAM and E-NPAM by using the reference values of adults. Therefore, these group-frailties can be compared with adult R-NPAM. Figure 3D shows that PAM display a group-frailty score higher than the one for adult R-NPAM. In contrast, E-NPAM have a group-frailty score of 0, even lower than that obtained in the group of adult R-NPAM. In addition, PAM exhibited a shorter lifespan than the group of R-NPAM and E-NPAM (p < .001), whereas the E-NPAM showed a longer lifespan than the group of R-NPAM (p < .01) (Figure 3B). The Individual Frailty Scores at Each Age Studied Correlate With Lifespan Given that the frailty profile is subjected to deficit accumulation and that the age-related deterioration of the physiological systems does not occur at the same rate in all subjects with the same chronological age, an individual frailty score was calculated for each mouse at each age by counting how many components of the test they failed. Again, depending on the age of the mouse, the corresponding reference values were applied as cut-offs. Thus, if a mouse failed three or more components out of the five that conform the test, it was considered as frail. If it failed one or two criteria, it was classified as prefrail, whereas if it did not fail any criteria it was considered as robust, according to the clinical classification for Fried’s Frailty Score (3). The results demonstrate that frailty within a group of mice of the same chronological age is very heterogeneous. Thus, at the late-adult age, within the group of R-NPAM, 10 per cent are frail, 20 per cent are prefrail, and 70 per cent are robust. Within the group of PAM, 40 per cent are frail, 40 per cent are prefrail, and only 20 per cent are robust, whereas within the group of E-NPAM all mice are robust (Figure 4A). Figure 4. View largeDownload slide (A) Frequencies of robust, prefrail, and frail mice in E-NPAM, R-NPAM, and PAM groups obtained using adult cut-off values as references. (B) Frequencies of robust, prefrail, and frail mice in adult, mature, and old R-NPAM groups obtained using adult, mature, and old cut-off values as references, respectively. Statistical differences were tested using Pearson’s chi-squared test. *: p < .05; **: p < .01. Figure 4. View largeDownload slide (A) Frequencies of robust, prefrail, and frail mice in E-NPAM, R-NPAM, and PAM groups obtained using adult cut-off values as references. (B) Frequencies of robust, prefrail, and frail mice in adult, mature, and old R-NPAM groups obtained using adult, mature, and old cut-off values as references, respectively. Statistical differences were tested using Pearson’s chi-squared test. *: p < .05; **: p < .01. In addition, individual frailty scores were also calculated at the mature and old ages by using as cut-off values the reference values at these ages. The results show that 44.5 per cent of the mature mice were robust, 44.5 per cent were prefrail, whereas 11 per cent were frail. Regarding the prevalence of frailty at the old age, it was found out that 50 per cent of the old mice were robust, 37.5 per cent were prefrail, whereas 12.5 per cent were considered frail (Figure 4B). Due to the observed heterogeneity regarding the individual frailty score of each mouse and given that the lifespan of each mouse was monitored individually, it was possible to investigate the relationship between the individual frailty score of a given mouse obtained at a given age, and its corresponding lifespan. Within the group of adult R-NPAM (Figure 5A), those mice which were prefrail and frail at the adult age lived significantly less time than their robust counterparts (p < .01, p < .001, respectively) and the frail mice even less than the prefrail ones (p < .05). Within the group of adult PAM (Figure 5B), those mice that were frail at the adult age lived significantly less than their robust counterparts (p < .05). Figure 5. View largeDownload slide Kaplan–Meier cumulative survival curves stratified in mice classified as robust, pre-frail and frail at the late-adult age (A), at the mature age (C), and at the old age (D) within the group of R-NPAM, as well as in adult PAM (B). Statistical differences between lifespans were tested using Kaplan–Meier log‐rank test. *: p < .05; **: p < .01; ***: p < .001. Figure 5. View largeDownload slide Kaplan–Meier cumulative survival curves stratified in mice classified as robust, pre-frail and frail at the late-adult age (A), at the mature age (C), and at the old age (D) within the group of R-NPAM, as well as in adult PAM (B). Statistical differences between lifespans were tested using Kaplan–Meier log‐rank test. *: p < .05; **: p < .01; ***: p < .001. In addition, the relationship between the individual frailty score of R-NPAM at the mature and old ages of each mouse (by using as cut-off values the reference values of their corresponding ages) and its respective lifespan was also studied. Within the group of mature R-NPAM (Figure 5C), those mice that were frail at the mature age lived significantly less than their robust (p < .01) and prefrail (p < .05) counterparts. In addition, within the group of old R-NPAM (Figure 5D), those that were frail at the old age lived significantly less than their robust counterparts (p < .05). Discussion It has been stated that in order to determine how to promote a healthy life expectancy in humans, common grounds between animal studies and clinical trials must be found (40). Several functions known to experience an age-related decline in humans can also be assessed in rodents. But many physiological tests that have been established in humans either do not exist or cannot be applied to aging experimental animals (40). Thus, the “Valencia Score” (17) becomes a useful tool for the quantification of frailty in mice, since it is noninvasive, simple and has the advantage of being comparable to the one that is routinely performed in humans, the Fried’s phenotype frailty score (3). However, in order to increase the potential applicability and translation of the “Valencia Score” results to humans, it is necessary to validate it in other mouse strains, as previously stated (15). Thus, the present study validates the use of the “Valencia Score” (17) for the quantification of frailty in outbred female ICR-CD1 mice. This is a novel finding giving that most of the research performed on frailty in mice has used inbred strains, which are less comparable to humans. Thus, data obtained in outbred strains, such as ICR-CD1, have a higher potential for clinical translation (41). The results show that as the mice aged, they showed a worse performance in the various tests that comprise the “Valencia Score”: running time, running speed, net grip strength, motor coordination, and weight loss, validating these criteria as good markers for the establishment of a frailty score in experimental animals. In addition, in the present study, frailty has been quantified in a model of prematurely aging mice, in order to shed light into the link between aging and frailty. Recently, frailty was quantified by the mouse FI (12), based on deficit accumulation, in the DBA/2J mouse strain, which is known to be short-lived. However, a higher frailty was only detected in males, compared with the frailty of the long-lived strain C57/BL6 (13). The quantification of frailty in the prematurely aging mice used in the present study has an important advantage over others, given that it is a natural model without genetic manipulation. PAM are just a fraction of the mice population which ages faster than their counterparts, and this makes the results more comparable to human subjects. The results demonstrate that the group of PAM, despite being adults, shows a higher frailty score to that obtained in adult R-NPAM, whereas the group of E-NPAM of the same age shows a frailty score of 0, even lower than the one obtained for R-NPAM. Thus, PAM are also prematurely frail. Moreover, the PAM group exhibited a shorter lifespan than the R-NPAM and E-NPAM groups, whereas E-NPAM displayed a longer lifespan than the R-NPAM group. Thus, these results demonstrate that there is a relationship between the frailty score, calculated by the “Valencia Score”, of a group of mice at the late-adult age and their respective lifespans. The use of the “Valencia Score” for quantification of frailty in a group as a whole has been shown to be useful in quantifying the effect of a given intervention, such as exercise, on frailty (17). However, quantification of individual frailty scores by the “Valencia Score” has not been performed. So far, the only studies that have calculated an individual frailty score for each mouse at different ages, throughout a longitudinal design, have used the FI based on deficit accumulation. The first one, proposed by Whitehead and colleagues (12) and further used by Kane and colleagues and Rockwood and colleagues (13,14), is a 31-item FI based on Signs of Clinical Deterioration in mice (hair loss, tremor, gait, etc.). This 31-item FI has been shown to be an indicator of biological age, given its relation to mortality. Although very complete, the translation of this FI to clinical practice seems difficult especially because the parameters used do not coincide with the accepted measures used clinically to define frailty in humans. The Physiological Frailty Index (PFI), proposed by Antoch and colleagues (15), is a 12-item Frailty Score that takes into account the variable grip strength plus another 11 biochemical ones (such as diastolic pressure, number of lymphocytes, and haematocrit). This score, although easier to implement, lacks relevant deficits that are predictors of frailty such as walking speed or motor coordination. It has been successfully applied to quantify the effect of dietary and pharmaceutical interventions on the FI. However, the link between individual frailty scores and respective lifespans was not studied, and the FI of a group was not always predictive of its lifespan. Therefore, the present study is the first one that has calculated an individual frailty score for each mouse at different ages, throughout a longitudinal design, by using the “Valencia Score” which is based on Fried’s phenotype frailty score. It is important to remark that in contrast to the frailty indices based on deficit accumulation, which take as reference values those from young adults, the “Valencia Score” requires the establishment of reference values (cut-off values) at each age. Thus, for the longitudinal design carried out in the present study, reference values were obtained at the late-adult, mature, and old ages, and frailty scores were therefore calculated at the same ages by using the age-matching values as cut-offs. Therefore, the prevalence of frail individuals at the adult, mature, and old ages was of 10, 12, and 11 per cent, respectively. This does not mean that frailty does not increase, given that they were calculated based on the performance of age-matching mice and therefore cannot be compared within themselves. In humans, the ability of a FI to predict variable vulnerability of individuals with the same chronological age has been referred to as their biological age (42). The “Valencia Score” developed for mice serves as an indicator of their biological age, given the high correlation between the individual frailty score of a mouse (independently of the age point at which it was established) and its respective lifespan. Thus, mice considered frail and prefrail showed a shorter lifespan compared to those that were robust at all ages studied. Moreover, those that were considered frail had a shorter lifespan than the prefrail ones. However, it should be taken into account that although biological age and frailty are closely related terms, they do not necessarily overlap. In fact, within the group of PAM, even though they are prematurely aged, differences within their individual frailty state were found. Thus, frail PAM, prefrail PAM, and robust PAM were observed, with the different frailty score having an impact on the lifespan achieved by each mouse. Noticeably, biological age seems to have a dominant role over the frailty score given that robust PAM have a shorter lifespan than robust R-NPAM. Thus, the individual frailty score, calculated by the “Valencia Score,” could be a useful analytic tool providing additional information in order to better define or classify individuals as prematurely aged. In summary, the “Valencia Score” for frailty has been shown to be useful in quantifying frailty in outbred female ICR-CD1 mice. The original contribution from the study is the demonstration that the “Valencia Score” can be applied for the calculation of an individual frailty score at the late-adult, mature, and old ages, acting as a sensitive predictor of lifespan. Moreover, the “Valencia Score” can be applied to quantify group-frailty in prematurely aging mice models in order to distinguish variable susceptibility to adverse events already at the adult age. The identification of frail adult mice opens up the possibility of carrying out long-term interventions, such as nutritional or exercise-based ones, starting at adult age as a preventive measure. These facts, together with the noninvasive and simple techniques used as well as the clinically relevant criteria that the “Valencia Score” takes into account, make it a good tool for obtaining frailty indices for experimental models. This allows longitudinal studies, to be performed in any laboratory. The results are likely to have potential applications in clinical settings. One limitation of the study is that reproducibility of the frailty scores obtained for each mouse was not investigated. Those calculations were only performed once in each mouse at each age point. As another consideration, it would have been interesting to obtain frailty scores on even older animals to explore the limits of frailty in mice by the “Valencia Score.” Funding This work was supported by grants of the Research group of UCM (910379), Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) (RD12/0043/0018) and (PI15/01787) from the ISCIII-FEDER of the European Union. J.V. and M.C.G.C. are supported by grants AICO/2016/076, SAF2016-75508-R, and ISCIII2012-RED-43-029 from RETICEF, CB16/10/00435 (CIBERFES), and PROMETEO2010/ 074. Conflict of interest statement None declared. References 1. 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Google Scholar Crossref Search ADS PubMed 41. von Zglinicki T , Varela Nieto I , Brites D , et al. Frailty in mouse ageing: a conceptual approach . Mech Ageing Dev . 2016 ; 160 : 34 – 40 . doi: 10.1016/j.mad.2016.07.004 . Google Scholar Crossref Search ADS PubMed 42. Mitnitski AB , Mogilner AJ , MacKnight C , Rockwood K . The mortality rate as a function of accumulated deficits in a frailty index . Mech Ageing Dev . 2002 ; 123 : 1457 – 1460 . doi: 10.1016/S0047-6374(02)00082-9 Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 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Abstract

Abstract The development of frailty scores suitable for mice and which resemble those used in the clinical scenario is of great importance to understand human frailty. The aim of the study was to determine an individual frailty score for each mouse at different ages and analyze the association between the frailty score and its lifespan. For this purpose, the “Valencia Score” for frailty was used. Thus, a longitudinal study in mice was performed analyzing weight loss, running time and speed, grip strength and motor coordination at the late-adult, mature and old ages (40, 56 and 80 weeks old, respectively). These parameters are equivalent to unintentional weight loss, poor endurance, slowness, weakness, and low activity level, respectively, in humans. A cut-off point was used to identify frail mice for each criterion. All the measurements were also performed on chronologically adult prematurely aging mice. The results show that by using the “Valencia Score” for frailty a prematurely aged phenotype can be identified even during the adulthood of animals. This opens up the possibility of carrying out preventive long-term interventions. Moreover, the individual frailty score of a given mouse at the late-adult, mature and old ages is shown to be a relevant predictor of its lifespan. Rate of aging, Longevity, Experimental models In the last decades, the aim of health care has switched from trying to live longer, to experiencing healthy aging. This is due to the current higher life expectancy being accompanied by an increase in disability rates and consequently, the lack of independence, autonomy, and well-being (1). Disability is often preceded by a state characterized by a diminished ability to respond to different stressors, which has been termed as frailty. In this regard, the current goal of improving healthy life expectancy is to act before disability arises by preventing or delaying the onset of frailty (2). Thus, frailty is a clinical geriatric syndrome defined by a diminished ability to restore homeostasis after any physical or mental damage, especially due to the absence of regulation of several physiological systems. The consequence of this being that when one individual faces minor stress situations, it results in outcomes like hospitalization, disability, and finally, death (3–7). Another important characteristic of frailty is that it is reversible, that is, it can be treated and even prevented. This is the reason why it has become necessary to determine which subjects are “frail” in the clinical scenario. In humans, there is a wide number of different scales to quantify frailty with different degrees of difficulty and clinical applicability (8). The two most commonly used ones in the clinical scenario are the phenotype frailty score and the frailty index (FI) based on deficit accumulation. The phenotype frailty score, developed by Fried and colleagues (3), takes into account five criteria: unintentional weight loss, slow walking speed, self-reported exhaustion, weakness and low physical activity. Individuals which meet three or more of these five criteria are classified as frail, individuals that meet one or two are classified as prefrail and those individuals with none are categorized as robust (3). In contrast, the FI, based on deficit accumulation, counts the number of potential health deficits of an individual and divides them by the total number of items measured (9). Another difference is that in the deficit accumulation approach, the reference values used are those of an adult, whereas in the phenotype frailty score, the reference values of the criteria are those corresponding to the age group studied. The development of frailty scores suitable for mice and which resemble those that are used in the clinical scenario has become an essential challenge in basic gerontological research (10), given that these would be useful tools to assess the effect of a given intervention in mice before translating it to humans. Thus, some frailty scores have been developed for the quantification of frailty in mice. Most of them have followed the deficit accumulation approach (9), by counting the presence or absence of a different set of deficits in mice (11–15). Following Fried’s phenotype frailty score (3), a frailty score has also been developed for the measurement of frailty in mice. It was first proposed by Liu and colleagues (16) and further developed by Gómez-Cabrera and colleagues (17). This last frailty score, which has been named the “Valencia Score” (17), adapts the same criteria to mice that are taken into account in humans (unintentional weight loss, endurance, slowness, weakness, and low activity level). Thus, weight loss, running time and speed, grip strength, and motor coordination were measured in mice. As it takes into account criteria that are closely related to the clinically accepted frailty score and it is simple to use; it may facilitate frailty research in animal models. Moreover, the results would have the potential to translate to clinical settings. Although some of the above-mentioned frailty indices, based on the deficit accumulation approach (14,15), have been shown to be predictive of mortality in mice, it is still not known if the “Valencia Score” would also be able to make this prediction. Most of the research performed on frailty has been focused on elderly people or mice. However, it is known that the age-related deterioration of the physiological systems does not occur at the same rate in all the individuals of a population with the same chronological age (18). Thus, subjects with the same chronological age can show large differences regarding health status and functional capacity and these interindividual differences can already be quantified at the adult age. This has been recently shown both in humans (19,20) and in mice (12,14,15). Even though some interventions initiated at the old age have been shown to be beneficial in decreasing the frailty status (13,17), the identification of frail individuals at the adult age would enable earlier interventions, which potentially could show more beneficial effects. However, in the “Valencia Score” for frailty, only 17 months old mice and older were studied (17) and it is still not known if it can be used to detect differences in the frailty status within younger mice. In agreement with the heterogeneity of the aging process, previous studies from our group have proposed a natural murine model of premature aging based on an inappropriate reactivity to stress. Thus, when mice are submitted to a simple T-maze test, those that need more time to explore it are classified as prematurely aging mice (PAM), whereas those that show an exceptional response to the maze, needing less time to explore it, are classified as exceptional non-PAM (E-NPAM). In addition, those mice that show an intermediate behavior, which constitute around 80 per cent of the population, are classified as regular non-PAM (R-NPAM). So far, it has been demonstrated that these PAM at the adult age show premature immunosenescence (21,22), an altered neurochemistry (23), higher anxiety-like behaviour (21,24), skeletal alterations (25), higher oxidative stress levels (21,26), and a shorter lifespan (27,28), compared with their counterpart E-NPAM of the same sex and chronological age (reviewed in Refs. 29 and 30). Hence, the first objective of the present work was to validate the “Valencia Score” in another sex and strain of mice (outbred female ICR-CD1) not previously studied, using a longitudinal study. Thus, a group-frailty score for each age (late-adult, mature, and old) was calculated. A group-frailty score was also calculated for E-NPAM and PAM at the late-adult age. The second objective was to determine an individual frailty score for each mouse at the late-adult, mature, and old ages (using as cut-off values the corresponding performance at each age) throughout a longitudinal study as well as to analyze the association between the frailty score of each mouse at a given age and its respective lifespan. For the longitudinal study, only R-NPAM were used. Materials and Methods Experimental Animals Female ICR/CD1 ex-reproductive mice (Mus musculus) of 32 ± 4 weeks were purchased from Janvier Labs (Germany) and placed in the Animal Facility at the Faculty of Biology (Universidad Complutense de Madrid, UCM). Mice were housed at 4–5 per cage. The average temperature in the Animal Facility was 22 ± 2°C, relative humidity was 60 per cent, and a 12/12 hour reversed light/dark cycle (lights on at 20:00h) was maintained to avoid circadian interferences. Mice were checked daily. Water and standard pellets (Panlab, Spain) were available ad libitum. Classification of mice into E-NPAM, R-NPAM, and PAM using a T-maze test After 1 week of acclimatization following the arrival of late-adult female ICR/CD1 mice (33 ± 4 weeks), the classification of these animals into PAM and NPAM was carried out. Previous experience in our laboratory has shown that 15 ± 5% of mice purchased behave as PAM and another 15 ± 5% behave as E-NPAM. Thus, 80 mice were submitted to the T-maze test in order to obtain enough mice from each type. The T-shaped maze is composed of three wooden arms (each 10 cm wide, 25 cm long, and 10 cm high) covered with black methacrylate. The floor consists of cylindrical aluminum rods that are 3 mm thick and arranged perpendicularly on the side walls. The test was carried out by holding the mouse by its tail and positioning it inside the base of the “T” with its head facing the end wall. Then, the time that each mouse took to cross the intersection of the “T” with both hind legs was measured. This test was performed once per week for a month, to distinguish PAM (which needed more than 10 seconds to cross the intersection at each test the four times) from the NPAM, as described previously (27,28). Depending on the behavior of NPAM, they were divided into E-NPAM (that required less than 10 seconds to cross the intersection at each test the four times) and R-NPAM (those that show an intermediate behavior spending less than 10 seconds sometimes and others more than 10 seconds). This test was always carried out under red light and between 09:00 and 11:00 hours in order to avoid circadian variations. Out of the 80 mice, 60 behaved as R-NPAM (75%), 10 as E-NPAM (12.5%), and 10 as PAM (12.5%). Not all mice were used for the study, only the following groups. One group of R-NPAM (n = 10) was used for the determination of the reference values for the “Valencia Score” at three different ages (40, 56, and 80 weeks, respectively). Another group of animals (n = 20), also R-NPAM, was used for the longitudinal study. The “Valencia Score” was carried out at the ages previously mentioned. Maximum lifespan in R-NPAM was 112 weeks with an average lifespan of 76 weeks. The 40 week old mice can be considered as late-adult mice, the 56 week old mice as mature mice, and the 80 week old animals as old mice. The “Valencia Score” test was also performed in 10 PAM and 10 E-NPAM at the late-adult age (40 ± 4 weeks) only. All the procedures were approved by the Experimental Animal Committee of UCM (Spain) and were in consonance with the European Community Council Directives 2010/63/EU of 22 September 2010 guidelines. Body Weight The body weights of the mice were recorded individually 1 month prior to and at the moment of carrying out the “Valencia Score” for frailty. When a given mouse lost more than 5 per cent of its weight compared with the previous month, it was considered frail for this criterion. This cut-off was selected in order to resemble the Fried’s phenotype frailty score for humans, which considers the loss of 5 per cent of body weight in the previous year as a frailty criterion (3). Motor Coordination Test The tight-rope test described by Miquel (31) and widely used by others (32) was used with the following modifications. Mice were placed in the middle of a bar of circular section (60cm long and 1.5cm diameter) and the test was considered successful if the mouse was able to reach either the end of the bar, or if it did not fall, during a period of 60 seconds in at least one out of the five consecutive trials. Incremental Treadmill Test Mice were submitted to an intensity treadmill test (Model LE8706, Panlab). The protocol of Davidson and coworkers (33) was used with the following modifications. Mice were placed on the treadmill at an initial speed of 6 m × minutes−1 for 6 minutes (warm-up period). After it, the treadmill band speed was increased by 2 m × minutes−1 every 2 minutes until the animals were exhausted. Exercise motivation was administered by the presence of an electric shock grid at the base of the treadmill. Exhaustion was defined as the refusal to run after three consecutive tail shocks. The maximum running time and running speed were recorded as measurements of their endurance and slowness, respectively. Grip Strength Test The Grip Strength Meter (Panlab. Harvard Apparatus) was used to measure the maximum force displayed by a mouse. Briefly, the grip strength meter was positioned horizontally and mice were held by the tail and lowered toward the apparatus. Animals were allowed to grasp the metal bar with their forelimbs and then they were pulled backwards in the horizontal plane (34). Maximum peak force of each mouse was automatically registered in grams-force. The test was performed five consecutive times. Since the weight influences the force of an animal, the net grip strength was adjusted by dividing the registered force by the weight of the animal. Data Analysis Analysis was performed with SPSS 21.0 (SPSS, Chicago, USA) software. Normality of the samples was checked by the Kolmogorov–Smirnov test and homogeneity of the variances was checked by the Levene test. Age-related differences in running time, running speed, and grip strength were studied using a one-way analysis of variance followed by post hoc analysis. The Tukey test was used for post hoc comparisons when variances were homogeneous, whereas Games–Howell analysis was used when variances were not homogeneous. Age-related differences regarding the tight-rope test and weight loss, as well as in the prevalence of frailty, were analyzed using Pearson’s chi-squared test. Differences in lifespan were investigated using the Kaplan–Meier test, with a minimum significance level (log rank, Mantel-Cox) set at p < .05. Two-sided p < .05 was considered the minimum level of significance. Results Group-Frailty, Quantified by the “Valencia Score”, Is Higher in PAM and Correlates With Lifespan The results show that both mature and old R-NPAM experience a decrease in the running time (endurance) as well as in the running speed (slowness) compared with when they are late-adults (p < .01 for mature mice; p < .001 for old mice). Old mice also experience a decrease in both components compared with when they are mature (p < .05 in running time; p < .01 in running speed) (Figure 1A and C). Regarding the premature aging model, chronologically adult PAM show lower running time and speed than E-NPAM (p < .05), whereas no statistically significant differences are found between PAM and late-adult R-NPAM (Figure 1B and D). Strikingly, E-NPAM show a higher running time than the late-adult R-NPAM (p < .05). Figure 1. View largeDownload slide Running time (A), running speed (C), grip strength (E), and net grip strength (G) values in R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The values corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B), (D), (F), and (H), respectively. These values were only compared with those of R-NPAM at the adult age. Statistical differences were tested using one-way ANOVA. a: p < .05; aa: p < .01; aaa: p < .001 with respect to the values in late-adult mice. b: p < .05; bb: p < .01 with respect to the values in mature individuals. c: p < .05; cc: p < .01 with respect to the values in E-NPAM. Figure 1. View largeDownload slide Running time (A), running speed (C), grip strength (E), and net grip strength (G) values in R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The values corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B), (D), (F), and (H), respectively. These values were only compared with those of R-NPAM at the adult age. Statistical differences were tested using one-way ANOVA. a: p < .05; aa: p < .01; aaa: p < .001 with respect to the values in late-adult mice. b: p < .05; bb: p < .01 with respect to the values in mature individuals. c: p < .05; cc: p < .01 with respect to the values in E-NPAM. Weakness is another key component of the diagnosis of clinical frailty (3). Thus, weakness of mice was investigated by measuring grip strength. As shown in Figure 1E, there are no age-related changes regarding grip strength in R-NPAM, whereas PAM show lower values than E-NPAM and late-adult R-NPAM (p < .05) (Figure 1F). There are many studies, all of them carried out in humans, which have found a positive correlation between grip strength and BMI or weight (35–37). Thus, in order to avoid differences in grip strength due to differences in body weight, a net grip strength variable was calculated by dividing the peak force registered for each animal by its weight. The results (Figure 1G) show that old R-NPAM experience a decrease regarding net grip strength compared with when they are late-adult (p < .05). In regard to the premature aging model, chronologically adult PAM show lower net grip strength than E-NPAM and R-NPAM (p < .01, p < .05, respectively) (Figure 1H). Strikingly, E-NPAM show higher net grip strength than late-adult R-NPAM (p < .001). The tight-rope test is a broadly used and extensively validated behavioral marker of aging (32,39). The modified protocol used in the present study was considered a good marker of motor coordination, which was quantified as the percentage of mice that succeeded in performing the test (17). In addition, unintentional weight loss was quantified as the percentage of mice that succeeded in not losing more than 5 per cent of their body weight compared with the previous month. The results (Figure 2A) show that old R-NPAM experience a decrease in the percentage of success in performing the tight-rope test in comparison to when they are late-adult (p < .01) and mature (p < .05). PAM have a lower percentage of success than E-NPAM, although nonstatistically significant differences are found (Figure 2B). Regarding weight loss, a tendency towards an age-related decrease in the ability to maintain body weight is observed, although no statistically significant differences are found at the different times of study in R-NPAM as well as in PAM (Figure 2C and D). Figure 2. View largeDownload slide Percentage of success in the tight-rope test (A) and unintentional weight loss (C) of R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The results corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B) and (D), respectively. Data regarding weight loss are expressed as percentage of mice that did not lose more than 5 per cent of their weight compared with the previous month. Statistical differences were tested using Pearson’s chi-squared test. aa: p < .01 with respect to the values in late-adult mice. b: p < .05 with respect to the values in mature individuals. Figure 2. View largeDownload slide Percentage of success in the tight-rope test (A) and unintentional weight loss (C) of R-NPAM at the late-adult (n = 20), mature (n = 18), and old (n = 8) ages (40, 56, and 80 weeks old, respectively). The results corresponding to PAM (n = 10) and E-NPAM (n = 10) are shown in (B) and (D), respectively. Data regarding weight loss are expressed as percentage of mice that did not lose more than 5 per cent of their weight compared with the previous month. Statistical differences were tested using Pearson’s chi-squared test. aa: p < .01 with respect to the values in late-adult mice. b: p < .05 with respect to the values in mature individuals. Based on the performance of mice at each age studied, reference values were obtained for each age by selecting the value of the 20th percentile for the variables running speed, running time, and net grip strength (Figure 3A). The variable net grip strength (grip strength/weight) was used instead of grip strength, given that the first showed a more marked age-related decrease (Figure 1E and G). Therefore, each mouse that ranked below the 20th percentile for any of these criteria was considered as a failure for that criterion. Regarding motor coordination and weight loss components, each mouse that failed to complete the tightrope test or lost more than 5 per cent of its body weight compared to the previous month, respectively, was considered as a failure for that criterion. Figure 3. View largeDownload slide (A) Reference values applied for each age investigated. (B) Kaplan–Meier cumulative survival curves of E-NPAM (n = 10), R-NPAM (n = 20), and PAM (n = 10). (C) Group-frailty score for adult, mature, and old R-NPAM obtained using, respectively, adult, mature, and old cut-off values as references. (D) Group-frailty score for chronologically adult E-NPAM and PAM obtained using adult cut-off values as references. Statistical differences between lifespans were tested using the Kaplan–Meier log‐rank test. **: p < .01; ***: p < .001. Figure 3. View largeDownload slide (A) Reference values applied for each age investigated. (B) Kaplan–Meier cumulative survival curves of E-NPAM (n = 10), R-NPAM (n = 20), and PAM (n = 10). (C) Group-frailty score for adult, mature, and old R-NPAM obtained using, respectively, adult, mature, and old cut-off values as references. (D) Group-frailty score for chronologically adult E-NPAM and PAM obtained using adult cut-off values as references. Statistical differences between lifespans were tested using the Kaplan–Meier log‐rank test. **: p < .01; ***: p < .001. For the calculation of the frailty score for each age group (late-adult, mature, and old R-NPAM), the corresponding age-matching reference values were applied. Each group-frailty score, expressed as a percentage, was obtained by dividing the total number of tests failed by the mice of each age, by the total number of tests performed by these mice. As can be seen in Figure 3C, the frailty score for late-adult R-NPAM is 15, for mature R-NPAM is 21, and for old R-NPAM is 25. These frailty scores cannot be compared among them, and they just represent an average frailty score for mice at these ages. In addition, following the same procedure described above, group-frailty was also calculated for adult PAM and E-NPAM by using the reference values of adults. Therefore, these group-frailties can be compared with adult R-NPAM. Figure 3D shows that PAM display a group-frailty score higher than the one for adult R-NPAM. In contrast, E-NPAM have a group-frailty score of 0, even lower than that obtained in the group of adult R-NPAM. In addition, PAM exhibited a shorter lifespan than the group of R-NPAM and E-NPAM (p < .001), whereas the E-NPAM showed a longer lifespan than the group of R-NPAM (p < .01) (Figure 3B). The Individual Frailty Scores at Each Age Studied Correlate With Lifespan Given that the frailty profile is subjected to deficit accumulation and that the age-related deterioration of the physiological systems does not occur at the same rate in all subjects with the same chronological age, an individual frailty score was calculated for each mouse at each age by counting how many components of the test they failed. Again, depending on the age of the mouse, the corresponding reference values were applied as cut-offs. Thus, if a mouse failed three or more components out of the five that conform the test, it was considered as frail. If it failed one or two criteria, it was classified as prefrail, whereas if it did not fail any criteria it was considered as robust, according to the clinical classification for Fried’s Frailty Score (3). The results demonstrate that frailty within a group of mice of the same chronological age is very heterogeneous. Thus, at the late-adult age, within the group of R-NPAM, 10 per cent are frail, 20 per cent are prefrail, and 70 per cent are robust. Within the group of PAM, 40 per cent are frail, 40 per cent are prefrail, and only 20 per cent are robust, whereas within the group of E-NPAM all mice are robust (Figure 4A). Figure 4. View largeDownload slide (A) Frequencies of robust, prefrail, and frail mice in E-NPAM, R-NPAM, and PAM groups obtained using adult cut-off values as references. (B) Frequencies of robust, prefrail, and frail mice in adult, mature, and old R-NPAM groups obtained using adult, mature, and old cut-off values as references, respectively. Statistical differences were tested using Pearson’s chi-squared test. *: p < .05; **: p < .01. Figure 4. View largeDownload slide (A) Frequencies of robust, prefrail, and frail mice in E-NPAM, R-NPAM, and PAM groups obtained using adult cut-off values as references. (B) Frequencies of robust, prefrail, and frail mice in adult, mature, and old R-NPAM groups obtained using adult, mature, and old cut-off values as references, respectively. Statistical differences were tested using Pearson’s chi-squared test. *: p < .05; **: p < .01. In addition, individual frailty scores were also calculated at the mature and old ages by using as cut-off values the reference values at these ages. The results show that 44.5 per cent of the mature mice were robust, 44.5 per cent were prefrail, whereas 11 per cent were frail. Regarding the prevalence of frailty at the old age, it was found out that 50 per cent of the old mice were robust, 37.5 per cent were prefrail, whereas 12.5 per cent were considered frail (Figure 4B). Due to the observed heterogeneity regarding the individual frailty score of each mouse and given that the lifespan of each mouse was monitored individually, it was possible to investigate the relationship between the individual frailty score of a given mouse obtained at a given age, and its corresponding lifespan. Within the group of adult R-NPAM (Figure 5A), those mice which were prefrail and frail at the adult age lived significantly less time than their robust counterparts (p < .01, p < .001, respectively) and the frail mice even less than the prefrail ones (p < .05). Within the group of adult PAM (Figure 5B), those mice that were frail at the adult age lived significantly less than their robust counterparts (p < .05). Figure 5. View largeDownload slide Kaplan–Meier cumulative survival curves stratified in mice classified as robust, pre-frail and frail at the late-adult age (A), at the mature age (C), and at the old age (D) within the group of R-NPAM, as well as in adult PAM (B). Statistical differences between lifespans were tested using Kaplan–Meier log‐rank test. *: p < .05; **: p < .01; ***: p < .001. Figure 5. View largeDownload slide Kaplan–Meier cumulative survival curves stratified in mice classified as robust, pre-frail and frail at the late-adult age (A), at the mature age (C), and at the old age (D) within the group of R-NPAM, as well as in adult PAM (B). Statistical differences between lifespans were tested using Kaplan–Meier log‐rank test. *: p < .05; **: p < .01; ***: p < .001. In addition, the relationship between the individual frailty score of R-NPAM at the mature and old ages of each mouse (by using as cut-off values the reference values of their corresponding ages) and its respective lifespan was also studied. Within the group of mature R-NPAM (Figure 5C), those mice that were frail at the mature age lived significantly less than their robust (p < .01) and prefrail (p < .05) counterparts. In addition, within the group of old R-NPAM (Figure 5D), those that were frail at the old age lived significantly less than their robust counterparts (p < .05). Discussion It has been stated that in order to determine how to promote a healthy life expectancy in humans, common grounds between animal studies and clinical trials must be found (40). Several functions known to experience an age-related decline in humans can also be assessed in rodents. But many physiological tests that have been established in humans either do not exist or cannot be applied to aging experimental animals (40). Thus, the “Valencia Score” (17) becomes a useful tool for the quantification of frailty in mice, since it is noninvasive, simple and has the advantage of being comparable to the one that is routinely performed in humans, the Fried’s phenotype frailty score (3). However, in order to increase the potential applicability and translation of the “Valencia Score” results to humans, it is necessary to validate it in other mouse strains, as previously stated (15). Thus, the present study validates the use of the “Valencia Score” (17) for the quantification of frailty in outbred female ICR-CD1 mice. This is a novel finding giving that most of the research performed on frailty in mice has used inbred strains, which are less comparable to humans. Thus, data obtained in outbred strains, such as ICR-CD1, have a higher potential for clinical translation (41). The results show that as the mice aged, they showed a worse performance in the various tests that comprise the “Valencia Score”: running time, running speed, net grip strength, motor coordination, and weight loss, validating these criteria as good markers for the establishment of a frailty score in experimental animals. In addition, in the present study, frailty has been quantified in a model of prematurely aging mice, in order to shed light into the link between aging and frailty. Recently, frailty was quantified by the mouse FI (12), based on deficit accumulation, in the DBA/2J mouse strain, which is known to be short-lived. However, a higher frailty was only detected in males, compared with the frailty of the long-lived strain C57/BL6 (13). The quantification of frailty in the prematurely aging mice used in the present study has an important advantage over others, given that it is a natural model without genetic manipulation. PAM are just a fraction of the mice population which ages faster than their counterparts, and this makes the results more comparable to human subjects. The results demonstrate that the group of PAM, despite being adults, shows a higher frailty score to that obtained in adult R-NPAM, whereas the group of E-NPAM of the same age shows a frailty score of 0, even lower than the one obtained for R-NPAM. Thus, PAM are also prematurely frail. Moreover, the PAM group exhibited a shorter lifespan than the R-NPAM and E-NPAM groups, whereas E-NPAM displayed a longer lifespan than the R-NPAM group. Thus, these results demonstrate that there is a relationship between the frailty score, calculated by the “Valencia Score”, of a group of mice at the late-adult age and their respective lifespans. The use of the “Valencia Score” for quantification of frailty in a group as a whole has been shown to be useful in quantifying the effect of a given intervention, such as exercise, on frailty (17). However, quantification of individual frailty scores by the “Valencia Score” has not been performed. So far, the only studies that have calculated an individual frailty score for each mouse at different ages, throughout a longitudinal design, have used the FI based on deficit accumulation. The first one, proposed by Whitehead and colleagues (12) and further used by Kane and colleagues and Rockwood and colleagues (13,14), is a 31-item FI based on Signs of Clinical Deterioration in mice (hair loss, tremor, gait, etc.). This 31-item FI has been shown to be an indicator of biological age, given its relation to mortality. Although very complete, the translation of this FI to clinical practice seems difficult especially because the parameters used do not coincide with the accepted measures used clinically to define frailty in humans. The Physiological Frailty Index (PFI), proposed by Antoch and colleagues (15), is a 12-item Frailty Score that takes into account the variable grip strength plus another 11 biochemical ones (such as diastolic pressure, number of lymphocytes, and haematocrit). This score, although easier to implement, lacks relevant deficits that are predictors of frailty such as walking speed or motor coordination. It has been successfully applied to quantify the effect of dietary and pharmaceutical interventions on the FI. However, the link between individual frailty scores and respective lifespans was not studied, and the FI of a group was not always predictive of its lifespan. Therefore, the present study is the first one that has calculated an individual frailty score for each mouse at different ages, throughout a longitudinal design, by using the “Valencia Score” which is based on Fried’s phenotype frailty score. It is important to remark that in contrast to the frailty indices based on deficit accumulation, which take as reference values those from young adults, the “Valencia Score” requires the establishment of reference values (cut-off values) at each age. Thus, for the longitudinal design carried out in the present study, reference values were obtained at the late-adult, mature, and old ages, and frailty scores were therefore calculated at the same ages by using the age-matching values as cut-offs. Therefore, the prevalence of frail individuals at the adult, mature, and old ages was of 10, 12, and 11 per cent, respectively. This does not mean that frailty does not increase, given that they were calculated based on the performance of age-matching mice and therefore cannot be compared within themselves. In humans, the ability of a FI to predict variable vulnerability of individuals with the same chronological age has been referred to as their biological age (42). The “Valencia Score” developed for mice serves as an indicator of their biological age, given the high correlation between the individual frailty score of a mouse (independently of the age point at which it was established) and its respective lifespan. Thus, mice considered frail and prefrail showed a shorter lifespan compared to those that were robust at all ages studied. Moreover, those that were considered frail had a shorter lifespan than the prefrail ones. However, it should be taken into account that although biological age and frailty are closely related terms, they do not necessarily overlap. In fact, within the group of PAM, even though they are prematurely aged, differences within their individual frailty state were found. Thus, frail PAM, prefrail PAM, and robust PAM were observed, with the different frailty score having an impact on the lifespan achieved by each mouse. Noticeably, biological age seems to have a dominant role over the frailty score given that robust PAM have a shorter lifespan than robust R-NPAM. Thus, the individual frailty score, calculated by the “Valencia Score,” could be a useful analytic tool providing additional information in order to better define or classify individuals as prematurely aged. In summary, the “Valencia Score” for frailty has been shown to be useful in quantifying frailty in outbred female ICR-CD1 mice. The original contribution from the study is the demonstration that the “Valencia Score” can be applied for the calculation of an individual frailty score at the late-adult, mature, and old ages, acting as a sensitive predictor of lifespan. Moreover, the “Valencia Score” can be applied to quantify group-frailty in prematurely aging mice models in order to distinguish variable susceptibility to adverse events already at the adult age. The identification of frail adult mice opens up the possibility of carrying out long-term interventions, such as nutritional or exercise-based ones, starting at adult age as a preventive measure. These facts, together with the noninvasive and simple techniques used as well as the clinically relevant criteria that the “Valencia Score” takes into account, make it a good tool for obtaining frailty indices for experimental models. This allows longitudinal studies, to be performed in any laboratory. The results are likely to have potential applications in clinical settings. One limitation of the study is that reproducibility of the frailty scores obtained for each mouse was not investigated. Those calculations were only performed once in each mouse at each age point. As another consideration, it would have been interesting to obtain frailty scores on even older animals to explore the limits of frailty in mice by the “Valencia Score.” Funding This work was supported by grants of the Research group of UCM (910379), Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) (RD12/0043/0018) and (PI15/01787) from the ISCIII-FEDER of the European Union. J.V. and M.C.G.C. are supported by grants AICO/2016/076, SAF2016-75508-R, and ISCIII2012-RED-43-029 from RETICEF, CB16/10/00435 (CIBERFES), and PROMETEO2010/ 074. Conflict of interest statement None declared. References 1. 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Journal

The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Sep 11, 2018

References

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