A toolbox for the longitudinal assessment of healthspan in aging mice

Abstract

The number of people aged over 65 is expected to double in the next 30 years. For many, living longer will mean spending more years with the burdens of chronic diseases such as Alzheimer's disease, cardiovascular disease, and diabetes. Although researchers have made rapid progress in developing geroprotective interventions that target mechanisms of aging and delay or prevent the onset of multiple concurrent age-related diseases, a lack of standardized techniques to assess healthspan in preclinical murine studies has resulted in reduced reproducibility and slow progress. To overcome this, major centers in Europe and the United States skilled in healthspan analysis came together to agree on a toolbox of techniques that can be used to consistently assess the healthspan of mice. Here, we describe the agreed toolbox, which contains protocols for echocardiography, novel object recognition, grip strength, rotarod, glucose tolerance test (GTT) and insulin tolerance test (ITT), body composition, and energy expenditure. The protocols can be performed longitudinally in the same mouse over a period of 4-6 weeks to test how candidate geroprotectors affect cardiac, cognitive, neuromuscular, and metabolic health.

Figure 1.

A graphic representation of the experimental design for healthspan assessment in response to a geroprotector of choice in C57BL6/J using the recommended toolbox. At each time point the multiple tests are performed over a period of 4-6 weeks, leaving those which may impact more on the animal at the end. In this case we recommend the following order, novel object recognition, frailty index, grip strength, cage top and hanging bar followed by rotarod, body composition, energy expenditure, glucose tolerance test (GTT), Insulin tolerance test (ITT) and echocardiography. Mo, Month.

Figure 2.. Representative images from high resolution echocardiography systems.

A) Representative image of M-mode image used for assessment of cardiac systolic function (AW = anterior wall, PW = posterior wall, LVEDD = left ventricular diastolic dimensions, LVESD = left ventricular systolic dimensions; arrows denote the thickness/dimension of interest for each variable). B) Representative image of pulsed-wave Doppler assessment of peak mitral valve inflow (E) using color Doppler guidance (IVRT = isovolumic relaxation time, IVCT = isovolumic contraction time, LVET = left ventricular ejection time). C) Representative image of tissue Doppler assessment of the septal mitral annulus velocity (e’) using the spectral velocity display. White arrows denote the point of measurement. Images are reproduced from Casaclang-Verzosa et al., 2017, J Vis Exp with permission. Animal procedures were performed following approval by the Mayo Clinic Institutional Animal Care and Use Committee.

Figure 3.. NOR memory enhancement in exercised male mice.

(A–B) NOR memory enhancement in exercised (RUN) vs. sedentary (SED) animals. Results represent as Discrimination index (DI). A DI score of >0.20 was set ad hoc to determine proper novel-object discrimination; statistically significant within-group differences were also considered between the training phase and the test phases. (A) Before exercising, mice were unable to discriminate the novel object in a difficult protocol. B) After 6 wk of physical exercise, exercised animals showed memory enhancement. A Mann–Whitney U test indicated that exercised male mice showed significantly higher DI scores than sedentary males in the short-term memory (STM) phase (U = 0, P = 0.021, r² = 0.67) and in the long-term memory (LTM) phase (U = 0, P = 0.02, r² = 0.67). A Friedman test revealed significant differences in the performance of exercised mice throughout the test (X² = 6.5, P = 0.039); post hoc analysis with Wilcoxon signed-rank tests showed significantly higher DI scores in both test phases (Z = −2.023, P = 0.043, r² = 0.41). For comparisons between independent groups, *P < 0.05; for intragroup differences (LTM vs training), +P < 0.05; tendencies 0.05 > #P < 0.09. Extreme values were removed from the analysis. SED, n = 4 biologically independent animals; RUN, n = 5 biologically independent animals. Figure adapted with permission from McGreevy et al, 2019, PNAS . The animal procedures were performed following approval by the Committee of Ethics and Animal Experimentation of the Cajal Institute, Ethics Committee of the CSIC, and the Animal Protection Area of the Ministry of Environment of the Community of Madrid. LTM, Long term memory, STM short term memory

Figure 4.. Assessment of muscle strength and neuromuscular function.

Physical function measurements in 20-month-old male mice treated with dasatinib and Quercetin (D+Q) once every 2 weeks (bi-weekly) for 4 months. (a) Grip strength, (b) hanging endurance, and (c) maximal walking speed on rotarod (relative to baseline) 4 months after drug initiation (n = 13 vehicle-treated and 20 D+Q-treated biologically independent animals per group, *P <0.05; n.s., not significant; Two-tailed Student’s t-tests). Results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. Figure is adapted from Xu et al., 2018, Nature Medicine with permission. Animal procedures were performed following approval by the Mayo Clinic Institutional Animal Care and Use Committee.

Figure 5.. Effects of dietary protein level on energy balance.

Metabolic chambers were used to assess A) Food Consumption, B) Spontaneous activity (as assessed by wheel running), C-E) Respiration and F) Energy Expenditure over a 24-hour period after approximately 8 weeks on the indicated diets (n= 9 biologically independent animals per group except for wheel count, 7% protein diet (n=8), two-tailed t-test, * = p < 0.05). Error bars represent standard error. Figure is adapted from Fontana, Cummings et al., 2016, Cell Reports with permission. Animal procedures were performed following approval by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital.

Figure 6.. Glucose and insulin tolerance tests.

A) Glucose tolerance test conducted on male C57BL/6J mice treated with either vehicle or with 2 mg/kg rapamycin (1x/day or 1x/5 days) for 2 weeks (n = 9 biologically independent animals per group; for GTT, Tukey–Kramer test following two-way repeated measures ANOVA, a = P < 0.05 vehicle vs rapamycin 1x/day, b = P < 0.05 vehicle vs. rapamycin 1x/5 days; for AUC, means with the same letter are not significantly different from each other, Tukey–Kramer test following one-way ANOVA, P < 0.05). Figure is adapted from Arriola Apelo et al., 2016, Aging Cell with permission. B) Insulin tolerance test on female C57BL/6J.Nia mice treated with either vehicle or rapamycin (2 mg/kg) once every 5 days for 8 weeks (n = 10 vehicle-treated and 9 rapamycin-treated biologically independent animals per group, two-tailed t-test). Test was performed 5 days after the last administration of either vehicle or rapamycin, at the conclusion of an overnight fast. Figure is adapted from Arriola Apelo et al., 2016, JGBS with permission. Error bars represent standard error. Animal procedures were performed following approval by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital.