Chinese Classics Recitation Training in Subjective Cognitive Decline

Unit of measure:

Whole-brain blood-oxygen-level-dependent (BOLD) signal change (%)

Detail Functional magnetic resonance imaging (fMRI) will be used to evaluate changes in task-related brain activation associated with the intervention. Whole-brain blood-oxygen-level-dependent (BOLD) signals will be analyzed using voxel-wise general linear modeling to identify activation patterns during task performance compared with resting baseline. First-level analyses will generate subject-specific contrast maps reflecting task-related neural activation. Group-level random-effects analyses will then be conducted to compare activation patterns between study groups and across time points. Statistical significance will be determined using a voxel-level threshold corrected for multiple comparisons using the false discovery rate (FDR). Effect sizes extracted from significant brain regions will be used for group-level comparisons.

Unit of Measure µV²/Hz (power spectral density)

Detail Resting electroencephalogram (EEG) recordings will be acquired using a standard 21-electrode montage positioned according to the International 10-20 system. Electrode impedance will be maintained below 10 kΩ, with a minimum sampling rate of 256 Hz. Recordings will be performed in a quiet, dimly lit environment with participants seated comfortably and instructed to remain awake with their eyes closed.

EEG preprocessing and analysis will be conducted using MNE-Python implemented in Python. The preprocessing pipeline will include re-referencing, resampling to 250 Hz, and band-pass filtering between 0.1 and 41 Hz. Artifact-free EEG signals will be segmented into non-overlapping 2-second epochs for subsequent spectral analysis. Quantitative EEG measures will be derived from the processed signals to characterize resting-state brain activity.

Outcome Measure Total score on the Mini-Mental State Examination (MMSE)

Unit of Measure points

Detail The MMSE is a widely used cognitive screening instrument assessing orientation, attention, memory, language, and visuospatial ability. Total score ranges from 0-30, with higher scores indicating better cognitive function.

Outcome Measure Total score on the Montreal Cognitive Assessment (MoCA)

Unit of Measure points

Detail The MoCA evaluates multiple cognitive domains including executive function, memory, attention, language, abstraction, and orientation. Total score ranges from 0-30, with higher scores indicating better cognitive performance.

Outcome Measure Total recall score on the Rey Auditory Verbal Learning Test (RAVLT)

Unit of Measure points

Detail The RAVLT assesses verbal learning and episodic memory through repeated recall of word lists across trials. Total recall score reflects the number of correctly recalled words, with higher scores indicating better memory performance.

Outcome Measure Interference completion time on the Stroop Color and Word Test

Unit of Measure seconds

Detail The Stroop test measures selective attention and cognitive control. The interference condition requires participants to name the ink color of incongruent color words. Lower completion time indicates better executive function.

Outcome Measure Total span score on the Digit Span Test

Unit of Measure points

Detail Digit Span evaluates auditory attention and working memory through forward and backward recall of number sequences. Higher scores indicate better working memory capacity.

Outcome Measure Total score on the Short Form (15 items) of the Boston Naming Test

Unit of Measure points

Detail The Boston Naming Test assesses confrontational naming ability. The short form contains 15 items, with total score ranging from 0-15, and higher scores indicating better language function.

Outcome Measure Global score on the Clinical Dementia Rating (CDR)

Unit of Measure scale score

Detail The CDR is a clinician-rated scale evaluating cognitive and functional performance in dementia. Global score ranges from 0 (no impairment) to 3 (severe dementia).

Outcome Measure Total score on the Hachinski Ischemic Score

Unit of Measure points

Detail The Hachinski Ischemic Score differentiates vascular from degenerative causes of cognitive impairment. Scores range from 0-18, with higher scores suggesting greater vascular contribution.

Outcome Measure Total score on the Geriatric Depression Scale (GDS)

Unit of Measure points

Detail The GDS evaluates depressive symptoms in older adults. Higher scores indicate more severe depressive symptoms.

Outcome Measure Total score on the Beck Anxiety Inventory (BAI)

Unit of Measure points

Detail The BAI measures the severity of anxiety symptoms. Total score ranges from 0-63, with higher scores indicating greater anxiety.

Outcome Measure Cognitive reserve index score on the Cognitive Reserve Index questionnaire

Unit of Measure index score

Detail The CRI-q quantifies lifetime cognitive reserve based on education, occupational complexity, and leisure activities. Higher index scores indicate greater cognitive reserve.

Unit of Measure genotype category

Detail Genomic DNA will be extracted from peripheral blood samples to determine the genotype of the APOE, a major genetic risk factor associated with cognitive decline and Alzheimer's disease. Participants will be categorized according to APOE genotype (e.g., ε2/ε2, ε2/ε3, ε3/ε3, ε3/ε4, ε4/ε4).

Unit of Measure IL-6: pg/mL TNF-α: pg/mL hsCRP: mg/dL Cortisol: µg/dL

Detail Peripheral venous blood samples will be collected after overnight fasting to quantify circulating inflammatory and stress-related biomarkers, including Interleukin-6, Tumor necrosis factor alpha, High-sensitivity C-reactive protein, and Cortisol. Blood samples will be collected in EDTA anticoagulant tubes and processed within 30 minutes by centrifugation at 2,000 × g for 10 minutes at 4 °C. Plasma will be aliquoted and stored at -80 °C until analysis.

Cytokine concentration HsCRP concentrations will be measured using high-sensitivity immunoassays to evaluate systemic low-grade inflammation. Plasma cortisol levels will be measured using validated immunoassay methods to assess hypothalamic-pituitary-adrenal (HPA) axis activity and physiological stress responses. Changes in these biomarkers will be analyzed to explore potential inflammatory and neuroendocrine mechanisms associated with the intervention.

Unit of Measure Shannon diversity index

Detail Fecal samples will be collected to evaluate changes in gut microbial diversity associated with the intervention. Microbial community composition will be characterized using full-length 16S rRNA gene sequencing, enabling species-level taxonomic identification of intestinal microorganisms. Alpha diversity will be quantified using the Shannon diversity index, which reflects both species richness and evenness within the microbial community. Changes in Shannon diversity between baseline and follow-up will be analyzed to assess intervention-related alterations in gut microbiota diversity.

Unit of Measure Relative abundance (%)

Detail Fecal microbiota composition will be analyzed using full-length 16S rRNA gene sequencing to determine the relative abundance of bacterial taxa at different taxonomic levels (e.g., phylum, genus, and species). Differential abundance analyses will be conducted to identify microbial taxa whose relative abundance changes over time or differs between study groups.

Unit of Measure Metabolite concentrations (µmol/g feces or relative abundance)

Detail Fecal metabolomic profiling will be performed using Liquid chromatography-tandem mass spectrometry to quantify metabolites associated with gut microbiota-host interactions. Metabolites of interest include short-chain fatty acids, bile acids, tryptophan pathway metabolites, neurotransmitter precursors, and inflammation-related metabolites. These analyses will be used to investigate potential metabolic pathways linking gut microbial activity with cognitive and neurophysiological outcomes.