The following protocol describes the procedures for collecting electroencephalographic (EEG), electrooculographic (EOG), electrocardiographic (ECG), neuropsychological, and structural magnetic resonance imaging (sMRI) data in patients with mild to moderate dementia due to Alzheimer’s disease (ADD – ADMCI), Parkinson’s disease (PDD – PDMCI), and Lewy bodies (LBD – LBMCI) and matched Cognitively Unimpaired Seniors.
The diagnosis of ADD and ADMCI is based on the criteria of the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR; American Psychiatric Association) and the National Institute of Neurological Disorders and Stroke–Alzheimer Disease and Related Disorders (NINCDS–ADRDA; Jack et al., 2011). Diagnostic criteria refer to the period when the diagnoses is performed. The global cognitive functioning is assessed using the Mini-Mental State Examination (MMSE; Folstein et al., 1975) for all the participants. We also administered the Cambridge Assessment of Memory and Cognition (CAMCOG; Roth et al. 1986) in some of them. For the evaluation of the non-cognitive symptoms and the specific affective and mood aspects, we used either the Geriatric Depression Scale (GDS; Yesavage et al., 1982) or the Cornell Depression Scale (Alexopoulos et al., 1988) and the Neuropsychiatric Inventory (NPI; Cummings et al., 1994). A specific clinical evaluation is made for ADD and ADMCI patients’ group to establish the severity of dementia using the Activity of Day Living (ADL) and the Instrumental Activities of Daily Living scale (IADL; Graf et al., 2008).
For the inclusion criteria of the ADMCI and ADD patients, we used those proposed by Albert et al., 2011: (i) the clinical diagnosis of AD is based on the above procedures; (ii) the determination of a worsening of episodic memory in the last six months and at least the deficits in another cognitive domain; (iii) the lack of patient autonomy in the activities of daily living (IADL<5); (iv) MMSE score ≤ 25; (v) the visual analysis of structural T1-weighted MRIs by local neuroradiologists with a report compatible with brain atrophy of the AD type.
The exclusion criteria for ADMCI and ADD patients are the presence of significant neuropsychiatric disorders, depression (detected with a GDS score higher than nine or with the Cornell Depression Score higher than 10), and other types or causes of dementia such as frontotemporal dementia (Rascovsky et al., 2011), vascular dementia diagnosed based on the National Institute of Neurological Disorders and Stroke and Association Internationale pour la Recherché et l’Enseignement en Neurosciences (NINDS-AIREN) working group (Gorelick et al., 2011), Parkinson’s disease (Gelb et al., 1999), Dementia with Lewy Bodies (McKeith et al., 2017), metabolic syndrome, nutritional deficits, tumors, epilepsy, etc. Exclusion criteria also include visual analysis of structural T2-weighted MRIs by local radiologists to exclude major cerebrovascular lesions and the chronic use of psychoactive drugs except for acetylcholinesterase inhibitors (all patients chronically took them) and NMDA receptor antagonists.
The diagnosis of PD is based on a standard clinical assessment of tremor, rigidity, and bradykinesia (Gelb et al., 1999). As measures of severity of a motor disability, the Hoehn and Yahr stage (Hoehn and Yahr, 1967) and the Unified Parkinson Disease Rating Scale-III (UPDRS III; Fahn and Elton, 1987) for extrapyramidal symptoms are used. Furthermore, the diagnosis of PDD is given to the patients with a history of dementia, preceded by a typical levodopa-responsive parkinsonian motor syndrome for at least 12 months and not related to other pathologic conditions than PD. The PDD is diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV-TR; American Psychiatric Association).
The clinical inclusion criteria for PDD status are (i) Clinical Dementia rating scale (CDR) ≥ 1; (ii) Activity of Day Living (ADL) < 3; (iii) Instrumental Activity of Day Living (IADL) < 3; (iv) MMSE score > 14 and < 24 (corrected); and (v) cognitive deficits in at least two domains extending z-scores of 1.5 in neuropsychological testing + progression of the cognitive symptoms after 12 months + IADL lost.
The exclusion criteria for PDD included the following forms of parkinsonism: (1) DLB (McKeith et al., 1996); (2) secondary parkinsonism, including drug-induced parkinsonism; (3) cerebrovascular parkinsonism; and (4) atypical parkinsonism with absent or minimal responses to antiparkinsonian drugs.
The clinical inclusion criteria for PDMCI status were (i) Clinical Dementia rating scale (CDR) ≤ 0.5; (ii) Activity of Day Living (ADL) > 3; (iii) Instrumental Activity of Day Living (IADL) > 3; (iv) MMSE score ≥ (corrected); and (v) cognitive deficits in at least one domain extending z-scores of 1.5 in neuropsychological testing + progression of the cognitive symptoms after 12 months.
The clinical exclusion criteria for PDMCI status were any neurological and psychiatric cause of major mental disorders (except cognitive deficits).
LBD and LBMCI were diagnosed according to the criteria for the diagnosis in research of the guidelines written by McKeith and colleagues (2017, 2020). The definition of LBD implies the presence of two core clinical manifestations (e.g., visual hallucinations, cognitive fluctuations, REM sleep behavior disorder, parkinsonism) with the dominant cognitive deficit preceding the motor deficits in the patient’s clinical history for at least one year. As measures of severity of motor disability for PDD and PDMCI patients, the Hoehn and Yahr stage and the Unified Parkinson Disease Rating Scale-III (UPDRS III; Goetz et al., 2008) for extrapyramidal symptoms were used. Alternatively, the diagnosis could be based on one core clinical manifestation and one indicative diagnostic biomarker, such as the positive biomarkers of dopaminergic deficits from dopamine transporter uptake from PET/SPECT, abnormal REM sleep behavior disorder revealed by polysomnography or abnormal cholinergic cardiac innervation using MIBG neuroimaging (McKeith et al., 2017, 2020).
The clinical inclusion criteria for LBD status were (i) Clinical Deterioration Rate (CDR) ≥ 1; (ii) ADL < 3, (iii) IADL < 3; (iv) MMSE score > 14 and < 24 (corrected); (v) cognitive deficits in at least two domains extending z-scores of 1.5 in neuropsychological testing + progression of the cognitive symptoms after 12 months + IADL lost.
The clinical inclusion criteria for LBMCI status were (i) CDR ≤ 0.5; (ii) ADL > 3, (iii) IADL > 3; (iv) MMSE score ≥ (corrected); (v) cognitive deficits in at least one domain extending z-scores of 1.5 in neuropsychological testing + progression of the cognitive symptoms after 12 months.
The clinical exclusion criteria for LBMCI and LBD status were any neurological and psychiatric cause of major mental disorders (except cognitive deficits).
The exclusion criteria for Cognitively Unimpaired Seniorsare (i) an MMSE score < 24 (Tombaugh et al., 1992); (ii) the presence of neurological or psychiatric diseases (previous or present), (iii) the presence of a condition of depression (detected with a GDS score higher than nine or with the Cornell Depression Score higher than 10), (iv) the use of chronic psychoactive drugs, and (v) significant chronic systemic illnesses (e.g., diabetes mellitus).
We use a neuropsychological battery to measure the main cognitive domains such as memory, language, executive function/attention, and visuo-construction abilities. In the specific, all patients should perform the following:
– the Digit Span Backward and Forward (Monaco et al., 2013) to examine the working memory;
– the Rey-Auditory Verbal Learning Test (RAVLT; Rey, 1958) to evaluate short- and medium-term episodic memory;
– the verbal fluency for letters and categories (Novelli, 1986) for examining respectively phonemic and semantic verbal fluencies, but they could also be used to assess executive functions (Shao et al., 2014);
– the Boston Naming test (Kaplan, 1983) for assessing language;
– the Trail Making Test (Part A and B; Reitan, 1955) for examining attention and executive functions, but it could also be used to measure visual scanning and working memory (Llinàs-Reglà et al., 2017).
– the Stroop test (Scarpina et al., 2017) to assess attention and executive functions, but it could also be used for examining processing speed, cognitive flexibility (Jensen and Rohwer, 1966), and working memory (Kane and Engle, 2003);
– the Benton Judgement of Line Orientation test (Calamia et al., 2011) to examine visuospatial judgment.
All rsEEG recordings should be performed if the participant reports having slept well the previous night and has no particular situation in the morning incompatible with the standard procedure (e.g. indisposition, etc.).
During recording, the participants sit on a comfortable armchair in a silent, dimly lit room. We ask them to remain awake, leave their mind to wander freely, and abstain from focusing on specific mental operations (e.g., recalling facts or semantic information, planning future behaviors, etc.).
For ADMCI, ADD, PDMCI, PDD, LBD and LBMCI participants, pharmacological administration (when planned) of routine drugs is postponed after the rsEEG recordings. More extended periods of suspension are not valid for obvious ethical reasons.
The rsEEG recordings (about 3-5 minutes in eyes-closed condition) is performed using 30 scalp exploring electrodes placed according to an augmented 10–20 system. We adopt a cephalic ground electrode while earlobe electrodes (A1 and A2) are used as an electric reference. All electrode impedances must be < 10 kΩ. Horizontal and vertical electrooculographic (EOG) standard bipolar channels will control eye and blinking movements.
The rsEEG and EOG data are amplified by a commercial CE-approved digital system, band-passed between 0.1 Hz and 70 Hz to undergo the analog-to-digital conversion and recorded at a sampling frequency of at least 256 Hz.
Figure 1. Scalp exploring electrodes (N = 30) for the resting-state eyes-closed electroencephalographic (rsEEG) recordings.These electrodes were placed over the scalp using an augmented 10–20 system. Typically (but not in all cases), a cephalic ground electrode was used, while earlobe electrodes (A1 and A2) were used as an electric reference.
When following the registration protocol above, it is important to remember that it is essential to avoid the following events (1) the onset of sleepiness (2) voluntary or involuntary movements or isometric muscle contraction. To maintain a constant level of vigilance, an operator must monitor the subject and EEG tracings online, verbally alerting the subject whenever signs of behavioral drowsiness (loss of muscle tone) and/or EEG (EEG slowing with K complexes and sleep spindles) occur. In the event of these signs, the experimenters talk to the subject until the EEG normalizes (marking the episode on the corresponding EEG traces and EEG recording notes).
All sMRI scans are performed with a high-resolution MRI scan are obtained by using techniques including axial T2-weighted dual-echo (TR: 3000 ms, TE: 96 ms, FOV: 240 mm, matrix: 256, slice thickness: 3 mm, gap: 0, NSA: 1), proton density-weighted dual-echo (TR: 3000 ms, TE: 12 ms, FOV: 240 mm, matrix: 256, slice thickness: 3, NSA: 1), and axial 3D T1-weighted TFE sequences (TR: 9 ms, TE: 4 ms, FOV: 240 mm, matrix: 256, slice thickness: 1 mm, NSA: 1). The only deviation from the ADNI protocol is the slice thickness of 1.0 mm instead of 1.2 mm.
The following Figure 1, 2, and 3 represent the workflow for developing EEG biomarkers in patients with aging-related neurodegenerative diseases. This workflow also reflects the “ontology” of those biomarkers and refers to the main data collection and analysis phases.