In this study, our goal was to use non-invasive neuroimaging to identify the impact of caloric intake on brain integrity over time. We used multi-metric imaging methods (PET/MRI/MRS) to determine CMRglc, brain metabolites and structural connectivity, and identified associations of their changes with cognitive functions.

Young control (5-6 mo), young calorie-restricted (5-6 mo), old control (18-20 mo), and old calorie-restricted C57BL/6 mice (18-20 mo) were obtained from the National Institute on Aging Caloric Restricted Colony. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky according to NIH guidelines.

Brain structural and metabolic integrity determination using MRI Brain structural and metabolic integrity were measured using a 7T Clinscan MR scanner. We used MRI-based diffusion tensor imaging (DTI) to measure fractional anisotropy (FA) in CC. Following DTI, brain metabolite levels were determined with proton (1H) MR spectroscopy (MRS) using a point-resolved spectroscopy sequence.

We used fluorodeoxyglucose (18FDG) positron emission tomography (PET) to measure CMRglc. The PET experiments were conducted at the University of Texas Health Science Center at San Antonio. The experimental procedure was approved by the IACUC of UTHSCSA.

The RAWM protocol consisted of a 2-day testing paradigm. A staggered training schedule was used, running the mice in cohorts of ten mice, while alternating the different cohorts through the trials over day 1 and day 2 of the test.

1-2 µl of blood sample was used to measure blood glucose level. Another 10 µl of blood sample was used for ketone bodies level measurement.

There are five key findings from the studies reported here. Firstly, there was an early onset of glucose reduction induced by CR. In contrast, increased ketone bodies were found in the young CR mice. These changes were age-independent — old CR mice had similar levels on those indices when compared to their young litter-mates. The findings are consistent with literature, indicating CR induced a metabolic shift from utilizing glucose to ketone bodies ^{1 2}.Secondly, we found that CR increases ATP production in young CR mice and preserves ATP production in old CR mice, relative to controls, based on the TCr data. Thirdly, we observed a preservation of white matter structural integrity with age in the CR mice. Fourthly, the mice fed with chronic CR diet preserved long-term memory. Finally, we found associations between glucose level, body weight, and lifespan. Taken together, we observed distinct patterns between normal aging and CR aging on brain functions. Normal aging shows reductions in brain glucose metabolism, white matter integrity, and long-term memory, resembling human brain aging. CR aging, in contrast, displays early onset changes in brain metabolism and preservations of energy production, white matter integrity, and long-term memory in aging mice.

Our findings suggest that the benefits found in the CR mice might be in part due to the early shift of energy metabolism caused by lower caloric intake. Because of reduced glucose availability, CR mice adapted to use ketone bodies metabolism at a very early age. These metabolic alterations remained stable with age. Moderate ketosis has been shown to have many beneficial properties for brain functions, including sustaining neuronal activities ³, preserving energy substrates ⁴, enhancing memory ^{5 6}, reducing insulin resistance, and alleviating damage caused by oxidative stress and hypoxia ^{4 7 8}. However, whether feeding ketogenic diet will mimic CR’s beneficial effects on brain functions remains an object of study ⁹.

It has to be pointed out that we used a long-lived rodent model in the present study to investigate CR effects. Recent studies have shown that the lifespan response to a single level of CR (e.g., 40% CR) varies widely in mice from different genetic backgrounds ¹⁰. It will be important in the future to use neuroimaging to determine if CR also has adverse effects on brain metabolic functions in rodent strains where deleterious effects on lifespan or cognitive functions are observed.

In conclusion, we successfully used non-invasive neuroimaging to identify CR effects on brain physiology in aging mice. Specifically, we found an early shift in brain metabolism in mice with low caloric intake, which was associated with preserved energy production, brain structural integrity, and long-term memory. Understanding nutritional effects on brain function may have profound implications in human aging and other age-related neurodegenerative disorders. Using multimodal neuroimaging methods, we will be in a position to identify effective nutritional interventions, and the treatment efficacy thereof, to slow sown brain aging and/or prevent dementia for humans.