The secondary outcome will be assessed at baseline and 6 and 18 months in the research lab (except for sleep studies, which will be conducted at the participant’s home) by personnel blinded to which group the participant was randomized. Due to the length of the study, outcomes measured at 6 and 18 months will likely be conducted by a different person who will be trained and validated on all study procedures they administer.

Apart from considering the overall cognitive composite score, specific domains hypothesized to be most sensitive to the aerobic exercise intervention (e.g., executive functioning and processing speed) will be analysed as secondary outcomes. The specific neuropsychological tests were chosen based on their relevance to ADRD and the component domains known to be affected by aerobic fitness training (executive functioning, complex attention, processing speed, and verbal memory [34, 6163]) (see Table Table33 for a complete listing of the individual neuropsychological tests that will be administered).

Neuropsychological tests administered for the Brain in Motion II trial

A CBF test will be conducted to investigate whether aerobic exercise enhances brain health (e.g. increase resting CBF) and, if yes, whether increased resting CBF due to training will be maintained over 1 year after the exercise intervention completion. Cerebrovascular responses to increases in arterial partial pressure of carbon dioxide (CO2) and to submaximal exercise (measures of cerebrovascular reserve) will be assessed using transcranial Doppler ultrasound. The experimental set-up and protocol used are described in detail in a previous publication from our laboratory [52]. Outcome measures for these tests include baseline CBF and cerebrovascular reserve (i.e., brain responses to CO2 and exercise), heart rate, blood pressure, and blood rheology (i.e., haematocrit, viscosity, and aggregation).

A protocol to assess maximal oxygen uptake (V̇O2max) will be performed to test the hypothesis that aerobic exercise will enhance aerobic fitness as measured by V̇O2max and that the V̇O2max improvement due to training will be maintained over 1 year after completion of the exercise intervention. The V̇O2max test will be conducted on a treadmill and will involve a ramp increase in workload aimed at reaching the subject’s V̇O2max within 8–12 min, according to ACSM recommendations [64]. Exercise testing will be completed in the Clinical and Translational Exercise Physiology Laboratory, Cumming School of Medicine, University of Calgary by certified exercise physiologists (Canadian Society of Exercise Physiology). The experimental set-up and the protocol used are similar to those described in previous studies from our lab (see [65]). Outcome measures for this test include V̇O2max, ventilatory thresholds, heart rate, and blood pressure.

Blood markers will be analysed to examine if they moderate the observed effects of aerobic exercise on cognitive outcomes. We hypothesize aerobic exercise will promote more favourable changes in the blood biomarkers than stretching-toning exercise, and changes in blood biomarkers in the aerobic exercise group will be associated with cognition enhancement. Sex steroid hormone status (estradiol, progesterone, testosterone, and sex hormone-binding globulin), lipids (cholesterol, high- and low-density lipoprotein, and triglycerides), thyroid (thyroid-stimulating hormone), renal (creatinine), hepatic (alanine aminotransferase and bilirubin), and cardiovascular disease markers (hsCRP) will be measured. Complete blood count will be quantified immediately after blood collection, while other markers will be assessed in batches after blood samples are centrifuged and frozen at −80 °C. Details of the blood volumes required and assays to be used (including reliability, validity, and coefficients of variation) are included in Table Table44.

Details of the blood volume required, assays to be used, intra-assay variability, and measuring range

Notes: All analyses will be carried out at Calgary Laboratory Services. At baseline (0 month), 27–30 mL of blood will be required for measurements of hormones, lipids, and screening markers of thyroid, kidney, hepatic function, and hsCRP (3 × 5 mL gold (SST tubes) + 3 × 4 mL lavender (EDTA tubes)). At the other time points (6 and 18 months), only 18–20 mL of blood will be required for measurements of hormones, lipid profiles, and hsCRP (2 × 5 mL gold (SST tubes) + 2 × 4 mL lavender (EDTA tubes))

Abbreviations: PST, plasma separator tube; SST, serum separator tube; EDTA, ethylenediaminetetra acetic acid in a tripotassium or disodium base; CV%, coefficient of variation; SHBG, sex hormone binding globulin; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; TSH, thyroid-stimulating hormone; CBC, complete blood count; ALT, alanine aminotransferase; CVD, cardiovascular disease; hsCRP, high-sensitivity C-reactive protein

DNA samples will be collected at baseline to test the hypothesis that genetic risk factors moderate exercise-related cognitive outcomes. We hypothesize aerobic exercise will promote greater cognitive enhancement in those with greater changes in neurotrophic factors (e.g., BDNF and IGF-1) compared to stretching-toning exercise. Genomic DNA will be obtained from buffy coat blood samples (Gentra Puregene Blood Kit; Qiagen, Venlo, Netherlands). DNA samples will be sent for polymerase chain reaction amplification and Sanger sequencing (BigDye v1.1 Cycle Sequencing Kit; Applied Biosystems, Foster City, CA) on ABI 3130XL Genetic Analyzer (Applied Biosystems). These techniques will be used to genotype selected genes that have shown to influence cognitive performance and that are associated with neuronal integrity. These genes include BDNF, APOE, IGF-1, catechol-O-methyl-transferase (COMT), angiotensin-converting enzyme (ACE), insulin-degrading enzyme (IDE), methylenetetrahydrofolate reductase (MTHFR), clusterin (CLU), complement component (3b/4b) receptor 1 (CR1), bridging integrator 1 (BIN1), phosphatidylinositol-binding clatherin assembly protein (PICALM), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and translocase of outer mitochondrial membrane (TOMM40).

Self-administered validated questionnaires will be used to quantify the role of additional lifestyle factors on cognitive functioning at baseline, and changes over the intervention and follow-up periods. Measures include changes in dietary intake, food frequency, supplement intake [66], physical activity [67, 68], motivation for physical activity [69], cognitive activities [62], mood changes [70, 71], social support [7274], social engagement [72, 75], and modifiable ADRD risk/protective factors [76], including attention deficit hyperactivity disorder (ADHD) [77]. These data will provide collective insights on the possible mechanisms by which our interventions improve cognitive functioning and help to prevent ADRD [76].

Brain MRI will be used to test the hypotheses that 6 months of aerobic training, but not stretching-toning training, is associated with the following: (1) increases in brain volume, specifically cortical grey and white matter volume, including the frontal lobes and cortical areas implicated in attention control and memory processes [27] and hippocampal volume [78]; (2) increases in MRI measured resting CBF [79]; (3) reduced progression of white matter hyperintensities of presumed vascular origin; and (4) increases in functional connectivity of the default mode network [80].

Neuroimaging data will be collected on the 3-T scanner (General Electric Discovery 750, GE Healthcare, USA). Our 37-min MRI protocol is built on the multi-site Alzheimer Disease Neuroimaging Initiative protocol [81]. Our protocol (see Table Table5)5) includes a high-resolution whole-brain 3D T1-weighted structural image, a T2-weighted fluid-attenuated inversion recovery (FLAIR) image to evaluate white matter hyperintensities, resting perfusion measured with arterial spin labelling (ASL), high angular resolution diffusion imaging to calculate mean diffusivity and fractional anisotropy and for tractography analysis, and resting-state blood oxygen level-dependent (BOLD) functional MRI for functional connectivity analyses. Further, we will generate cerebrovascular reactivity maps from BOLD and ASL time series acquired during hypercapnia [8287]. It is expected that 60–70% of participants will consent to the MRI component of the study; however, participants with contraindication for an MRI exam will not be included in this part of the study. Therefore, an additional 10–20% non-completion rate is expected for participants who do consent to an MRI.

MRI acquisition parameters

Abbreviations: FLAIR, weighted fluid attenuated inversion recovery; BOLD, resting-state blood oxygen level dependent; CVR, cerebrovascular reactivity; pCASL, pseudo-continuous arterial spin labelling

We will test whether the beneficial effect of aerobic exercise on cognitive functioning is modulated by an improvement in sleep quality. We will use three complementary modalities to monitor sleep quality. First, we will apply the Pittsburgh Sleep Quality Index (PSQI) Questionnaire [88], which assesses the participants’ quality of sleep during the previous month. Second, we will measure inactivity as a proxy for sleep using actigraphy [89]. Finally, we will analyse sleep quality through an in-home-based overnight level two polysomnography (Embletta MPR PG, Natus Medical Inc., Pleasanton, CA) and an ST1 proxy unit.

To estimate the cost-utility of our aerobic exercise intervention, we will assess costs associated with the intervention itself (e.g., recreational facility membership, personal trainer time, equipment), physical activity engaged in during the maintenance phase, and healthcare resource utilization. Using provincial administrative databases, costs of pharmaceuticals, primary care physician visits, emergency room visits, and hospitalizations will be calculated. We will use the EuroQoL Five-Dimension Five-Level Quality of Life Scale (EQ-5D-5L [9092];) to measure the quality of life. The scores will be translated into utilities using the Canadian social value set [93].

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