SUCCESS CRITERIA
Primary Success Criteria (Hierarchical)
(1) Network Hyperconnectivity Reduction - Mandatory Threshold: Achieve statistically significant (p<0.05, two-tailed t-test with Bonferroni multiple comparisons correction across 12 DMN seed regions) between-group difference in default mode network functional connectivity by week 8. Specific target: posterior cingulate cortex - medial prefrontal cortex Pearson correlation coefficient reduction of ≥25% in intervention group (absolute reduction ≥0.15 correlation units) compared to controls with <8% change. This criterion must be met to proceed with claim of efficacy; failure requires protocol re-evaluation. Validation criterion: effect size Cohen's d ≥0.85 for between-group comparison.
(2) Tau Propagation Attenuation - Mandatory Threshold: Demonstrate ≥40% reduction in phosphorylated tau (pTau181 or pTau217) concentration in hippocampal CSF (microdialysate) measured at weeks 8-12 in intervention group compared to control group baseline-adjusted trajectories (p<0.01, repeated-measures ANOVA with group×time interaction, corrected for multiple comparisons across 4 timepoints). This must be corroborated by immunohistochemistry showing ≥40% reduction in hyperphosphorylated tau (AT8+ or pS396-tau+) burden in CA1-CA3 pyramidal layer relative to controls (quantified by blinded stereological point-counting across 3 coronal sections per animal, 8 animals per group minimum). Combined effect size must exceed d ≥0.90 across biomarkers.
(3) Neuronal Integrity Preservation - Mandatory Threshold: Maintain neuronal density loss to <10% in intervention group compared to age-matched AD transgenic controls at week 12 endpoint. Criterion operationalized as: (a) NeuN+ cell counts in hippocampus (CA1-CA3 combined) and medial prefrontal cortex (layers II/III) decline ≤8% from baseline to week 12 in intervention group versus ≥18% decline in controls (p=0.006, independent-samples t-test), (b) active caspase-3+ apoptotic cells reduced by ≥50% in intervention versus controls, (c) electron microscopy ultrastructure analysis showing preservation of synaptic integrity (synaptic cleft width 18-22 nm, normal presynaptic vesicle density ≥30 vesicles/100 nm² in ≥85% of quantified synapses in intervention group versus <65% in controls).
Secondary Success Criteria (Supporting Evidence)
(4) Cognitive Performance Improvement: Morris water maze escape latency improvement of ≥20% in intervention group by week 8 (baseline 35±8 seconds to ≤28±6 seconds) with maintained performance through week 16, compared to progressive deterioration in controls (baseline 36±7 to 48±9 seconds, p=0.008, two-way repeated-measures ANOVA). Object recognition discrimination index in intervention group improves to ≥0.55 (chance=0.50) by week 8, versus <0.52 in controls.
(5) Sleep Architecture Normalization: Increase spontaneous sleep spindle density by ≥40% in intervention group (baseline 8.2±1.3 to ≥11.5 spindles/minute NREM sleep) measured via electrocorticography, with effect maintained at week 16 (p<0.01). Spindle-fast oscillation coupling strength increases ≥30% in intervention group, quantified via wavelet-based time-frequency analysis.
(6) Cross-Regional Coherence Enhancement: Hippocampal-cortical theta coherence (4-8 Hz) increases ≥30% in intervention group versus <10% in controls (p=0.003, interaction effect). Gamma phase-locking value between hippocampus and prefrontal cortex increases ≥25% during active exploration in intervention group.
(7) Microglial Phenotype Shift: Ramified microglial morphology restored in ≥65% of Iba1+ cells in intervention group versus <40% in controls (p=0.004). Pro-inflammatory/anti-inflammatory cytokine ratio (IL-6+TNF-α/IL-10) decreases ≥35% in intervention group CSF by week 8.
Failure Criteria - Protocol Termination Thresholds
(8) Safety Endpoint: Intervention discontinued if ≥2 animals experience seizure activity, unexplained mortality, or ≥20% body weight loss. Any electrode-related infection or intracranial hemorrhage detected via MRI mandates individual animal removal and device re-assessment. Study halted if adverse event frequency exceeds 15% in intervention group.
(9) Efficacy Futility: If primary criterion #1 (network connectivity reduction) shows between-group p-value >0.20 at week 8 interim analysis (n=12/group), protocol undergoes revision or termination as per pre-specified futility rules. If tau reduction criterion #2 shows <15% reduction in intervention group by week 8, alternative stimulation parameters must be tested with formal amendment.