The NutBrain Study aims to understand the biological mechanisms through which diet influences cognitive disorders with a special focus on the impact of nutrition on gut microbiota and brain characteristics, by applying a novel multi-level approach that integrates traditional epidemiological methods with neuroimaging and gut microbiota profiling. Aims of the NutBrain study are: i) to estimate the occurrence of MCI and other cognitive disorders in community-dwelling older people aged 65 + years; ii) to investigate the association between lifestyle habits and cognitive ageing outcomes; iii) to explore the role of diet, in modulating the gut microbiota composition, which in turn impacts on brain structures and functions as well.

The NutBrain Study is an ongoing population-based cohort study promoted by the Institute of Biomedical Technologies of the National Research Council (ITB-CNR, Segrate, Italy) and the Hospital IRCCS Fondazione Mondino (Pavia, Italy). The NutBrain Study is structured in three phases:

T=1 Participant's recruitment. The study is performed in two sites in the outskirt of North-Milan: Bollate and Baranzate. A random sample is drawn from the official register of residents in the two municipalities. Inclusion criteria are: attending a medical appointment in the research facility, living at home in one of the two municipalities, and being 65+ years. Eligible population is contacted by means of a letter of invitation, in which people are invited to contact the ITB-CNR by phone to schedule the first visit during which he/she is asked to bring last instrumental and clinical exams prescribed by their general practitioner. A comprehensive communication campaign is implemented to publicize and promote the project as well as to foster recruitment. To ensure a high enrolment rate, the Mayors of the two municipalities have been actively involved since the earliest planning stages of the recruitment and have granted patronage to the project. Additionally, the study is promoted and advertised at the community level through different channels: by printing brochures and setting posters in senior recreation centres, shops, pharmacies, medical doctors office, and patients associations; by articles and advertisements in local newspapers, institution websites, as well as in social media (e.g. Facebook, https://www.facebook.com/TheNutBrainStudy/). The NutBrain Study has a website (www.nutbrain.it) where potential participants are directed for further information and can register interest to participate. In addition, to raise awareness, several public events describing the objectives of the proposed study are organized for recruiting eligible people.

T=2 Screening evaluation. Participants are visited at the research facility in their residence town (arranging home visits for disabled individuals) by a trained team. Informed consent form is completed at the research facility prior to data collection. In those individuals without capacity to give full informed consent, proxy consent is collected from relatives or caregivers. This 2-hours interview includes a face-to-face administration of a neuropsychological battery of tests and questionnaires to inquire about socio-demographic, occupational, and social-economic data, education, medical conditions and drug use, lifestyle habits, functional status, and dietary behaviours. At the end of the visit, a stool sampling kit, consisting of a sterile faeces container with instructions explaining the procedure for the stool sample collection, and a 3-day food diary is provided to each participant. The first visit serves to screen each participant for cognitive functions and for identifying inclusion/exclusion criteria in a more comprehensive manner.

T=3 Clinical evaluation. Individuals suspected to have cognitive impairment at the screening neuropsychological tests undergo a clinical examination. Participants are transported from their own home to the Hospital using a private transport service, whose cost is covered entirely by the study. The hospital visit includes a standard neurological examination performed by a neurologist and a 3T Magnetic Resonance Imaging (MRI) scanning. At this stage, blood and stool samples are collected from each participant and stored in the biorepository at the Hospital until processing. For each subject with a clinical diagnosis of MCI, two cognitively intact controls of the same sex and of the closest date of birth are enrolled and visited at the Hospital (case-control design), undergoing the same protocol as MCI individuals.

Date security Data are handled, monitored, computerised, and stored in accordance with the European General Data Protection Regulation (EU) 2016/679 (GDPR) (https://gdpr-info.eu/). All study records, including the consent forms, are kept in a locked filing cabinet at the ITB-CNR where the file server for data storage is located. The file server is firewalled within the ITB-CNR intranet. For privacy and security, a password granted only to the server administrator is required to access to the database. The data forms are double checked for missing data and inconsistencies. Quality of the database entered data is monitored by checking entry for a random sample of participants. Data transfer is protected by means of crypting/decrypting policy and password protection. In the final dataset a unique key identifies each subject to guarantee anonymity. Personal data are regarded as strictly confidential and removed before the exportation procedure. Security of data is guarantee via automatic backups.