GoKawiilMice
C57BL/6 mice (female, aged 20 months) were obtained from the National Institute on Aging. Mice were acclimatized for a week after arrival at the facility and before starting experimental treatments or baseline measurements. As described previously12, group housing is standard practice for calorie-restriction studies. Mice were group housed under a 12 h–12 h light–dark cycle at 20–26 °C and 30–70% humidity. Control and semaglutide-treated groups had ad libitum access to water and standard laboratory chow diet (LabDiet, Rodent Diet 5053). Calorie-restricted mice were provided with unlimited access to water and measured amount of food daily. Competition for food was minimized by placing food directly into the bottom of the cage, allowing individual mice to get a pellet. The degree of restriction was based on the mean reduction in food intake induced by semaglutide and not adjusted over time. Mice received daily subcutaneous injection of 10 nmol per kg semaglutide or an equal volume of saline. For the lifespan study, 39 mice received saline and 40 mice received semaglutide for the duration of the lifespan. For physiological, molecular and cellular studies, a separate cohort was treated for 3 months. Ten pairs for physiological assessments, five pairs for neural stem cell studies requiring tissue fixation, and six pairs for other molecular and cellular analyses. For longitudinal study, a separate cohort was treated with saline, semaglutide or calorie restriction for 5 months (10 mice per group). Measurements were assessed at the baseline, after 2 or 4 months of treatment.
For subcutaneous injection, we followed the standard operating protocol established by the animal care committee at the University of California, Berkeley. In brief, subcutaneous injections were performed daily using a 28-gauge needle in the dorsal subcutaneous region. To reduce discomfort, a new needle was used for each animal. To minimize local tissue irritation and the risk of chronic inflammation, the operator systematically rotated injection sites across the dorsal surface, ensuring no single site was used repeatedly in consecutive days. Animals were monitored daily for local reactions, including erythema, swelling, ulceration or tissue damage. No adverse effects were observed at any timepoint. On-site veterinarians were overseeing health status checks. All animal procedures were performed in accordance with the animal care committee at the University of California, Berkeley.
Lifespan
Mouse lifespan was determined according to previous studies12. In brief, mice were examined daily for survival and signs of illness. Mice found dead were noted at daily inspection. Severely moribund mice were killed and recorded. A mouse was considered severely moribund if it showed one of the following clinical signs: (1) tumour formation; (2) untreatable wounds; (3) untreatable skin ulceration or abscesses; (4) severe rectal prolapse; (5) inability to eat or drink or any condition that interferes with the ability to eat or drink, lack of response after stimulation; (6) severe dehydration; (7) respiratory distress, agonal breathing, cyanosis; (8) paralysis/paresis; (9) uncontrollable haemorrhage; (10) unrelievable, progressive hypothermia; (11) body condition score <2 out of 5. Following established practice12, both mice euthanized or found dead were represented as deaths in the survival curves. No mice were removed from the study for reasons unrelated to age-associated decline and censored in the survival analysis.
Body composition
Body composition of mice was measured using an EchoMRI-100V Body Composition Analyzer (EchoMRI). Fat mass and lean mass data were collected.
Open-field test
As described previously33, mice were acclimatized to the testing room under normal light for 1 h before testing. Mice were placed at the centre of a plastic chamber (50 × 50 cm) and allowed to move freely for 15 min. Mouse activity was recorded using a digital video camera and analysed with EthoVision (XT14, Noldus Information Technology). The central zone of the chamber was defined by the distance to the wall equivalent to the length of the mouse.
Elevated plus maze
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