{"artifact":{"id":"experiment_proposal-afa14449-d6f1-4587-8756-49323c4ea1b5","artifact_type":"experiment_proposal","entity_ids":"[\"h-SDA-2026-04-26-gap-20260426-001521-01-plasma-nfl-elevation-secondary-to-bbb-associated-873d04c826\"]","title":"Experiment Proposal: h-SDA-2026-04-26-gap-20260426-001521-01-plasma-nfl-elevation-secondary-to-bbb-associated-873d04c826","quality_score":0.75,"created_by":"forge_experiment_proposal_generator","provenance_chain":"[]","content_hash":"3bcb72b76247afccb28ecadd57d92e250ede4dc381b6b7283695a245a834352f","metadata":{"citations":["38735950","37875471","33580742","29377008","30280653","22048062"],"generated_at":"2026-04-28T19:59:47.148253+00:00","intervention":"Acute BBB modulation via intraperitoneal lipopolysaccharide (LPS, 1 mg/kg) or sterile saline (vehicle) at 3 timepoints: 24h, 72h, and 7 days post-injection. BBB integrity assessed via gadolinium-enhanced MRI at 2h post-injection and Evans blue extravasation at 24h. Plasma and CSF sampling at baseline (pre-injection) and each timepoint via submandibular bleed and cisterna magna collection, respectively.","key_controls":["Age-matched C57BL/6J WT mice treated with LPS (negative control for neurodegeneration-related NfL elevation)","Age-matched 5xFAD mice treated with sterile saline (vehicle control for LPS effects)","Age-matched WT mice treated with sterile saline (baseline reference)","Technical control: pooled mouse plasma quality control sample included on every Simoa plate (inter-assay CV < 10%)","BBB integrity control: gadolinium-enhanced MRI in subset (n=6 per group) confirming LPS-induced permeability","Sex-matched comparisons within each genotype group"],"model_system":"C57BL/6J and 5xFAD transgenic mice (Alzheimer's disease model with amyloid pathology and progressive BBB dysfunction). Both sexes, 3 months and 9 months of age (pre-symptomatic and symptomatic stages). n=20 per group (sex-balanced: 10 male, 10 female) for longitudinal cohort. Genotype: 5xFAD (APP Swedish, Florida, London mutations + PSEN1 M146L, L286V) on C57BL/6J background. Littermate wild-type C57BL/6J mice as controls.","hypothesis_id":"h-SDA-2026-04-26-gap-20260426-001521-01-plasma-nfl-elevation-secondary-to-bbb-associated-873d04c826","timeline_weeks":18,"_schema_version":1,"null_hypothesis":"Plasma NfL levels do not correlate with BBB permeability, and the plasma:CSF NfL ratio is not elevated in subjects with evidence of BBB disruption compared to those with intact BBB, indicating that plasma NfL does not serve as a sensitive indicator of BBB permeability-augmented neurodegeneration.","primary_readout":"Plasma NfL concentration quantified by Simoa NF-Light Advantage assay (Quanterix) in pg/mL, measured in duplicate. Longitudinal plasma:CSF NfL ratio calculated at each timepoint.","estimated_cost_usd":128500.0,"secondary_readouts":["CSF NfL concentration by Simoa (parallel measurement to plasma)","BBB permeability markers: plasma soluble PDGFR-beta (sPDGFR-beta), CSF/serum albumin ratio, gadolinium enhancement volume on T1-weighted MRI","Plasma GFAP (astrocyte activation marker) by Simoa","Brain atrophy measurement via 9.4T MRI voxel-based morphometry at endpoint","Cortical and hippocampal NfL immunoreactivity by immunohistochemistry at endpoint","A beta plaque burden (thioflavin S) and astrogliosis (GFAP) at endpoint","Plasma IL-6 and TNF-alpha (systemic inflammation markers) by ELISA"],"expected_effect_size":"Cohen's d = 0.85 for plasma NfL elevation in 5xFAD vs. WT at 9 months (based on pilot data from PMID 38735950 indicating ~2.5-fold elevation). Power = 0.85 at alpha = 0.05 (two-tailed). For the primary comparison (plasma:CSF NfL ratio post-LPS), expecting Cohen's d = 1.2 based on literature precedent for acute BBB modulation effects. Sample size calculation assumes 20% attrition (death, sampling failure) over longitudinal timepoints.","statistical_approach":"Linear mixed-effects model with subject as random intercept for longitudinal plasma NfL and plasma:CSF ratio. Fixed effects: genotype (5xFAD vs. WT), time, treatment (LPS vs. saline), sex, and genotype×time interaction. Post-hoc pairwise comparisons with Bonferroni correction for multiple testing (primary comparison: 5xFAD-LPS vs. 5xFAD-saline at 24h timepoint). Significance threshold: alpha = 0.05. Repeated measures ANOVA as sensitivity analysis. Normality assessed via Shapiro-Wilk; non-parametric alternatives (Friedman test) if assumptions violated.","sample_size_rationale":"Power analysis (G*Power 3.1) based on two-way ANOVA for genotype×treatment interaction with effect size f = 0.40 (medium-large). With n=16 per group (accounting for 20% attrition from initial n=20), power = 0.85 at alpha = 0.05. This exceeds the minimum recommended power of 0.80 and is consistent with published biomarker studies in 5xFAD mice (PMID 33580742 methodology reference for blood-based biomarker detection).","falsification_criterion":"The hypothesis is falsified if: (1) plasma NfL levels do not differ significantly between 5xFAD and WT mice at 9 months (mixed-effects model p > 0.05), OR (2) the plasma:CSF NfL ratio is not elevated in the LPS-treated 5xFAD group compared to saline-treated 5xFAD controls (post-hoc Bonferroni p > 0.05), OR (3) plasma NfL elevation correlates inversely with BBB permeability markers (indicating NfL entry into plasma is barrier-restricted, not barrier-enhanced; Pearson r < 0 with p < 0.05)."},"created_at":"2026-04-28T12:59:47.160067-07:00","updated_at":"2026-04-28T12:59:47.160067-07:00","version_number":3,"parent_version_id":null,"version_tag":null,"changelog":null,"is_latest":1,"lifecycle_state":"active","superseded_by":null,"deprecated_at":null,"deprecated_reason":null,"dependencies":null,"market_price":0.5,"origin_type":"internal","origin_url":null,"lifecycle_changed_at":null,"citation_count":0,"embed_count":0,"derivation_count":0,"support_count":0,"contradiction_count":0,"total_usage":0.0,"usage_score":0.5,"usage_computed_at":null,"quality_status":null,"contributors":[],"answers_question_ids":null,"deprecated_reason_detail":null,"deprecated_reason_code":null,"commit_sha":null,"commit_submodule":null,"last_mutated_at":"2026-05-16T14:51:34.657673-07:00","disputed_at":null,"gap_id":null,"mission_id":null,"intrinsic_priority":null,"effective_priority":null,"artifact_id":"24c416c0-72cc-473d-a191-50d42068808c","artifact_dir":null,"primary_filename":null,"accessory_filenames":null,"folder_layout_version":1,"migrated_to_folder_at":null,"hypothesis_id":null,"authorship":{"kind":"human","contributors":[{"role":"author","actor_ref":"forge_experiment_proposal_generator"}]},"epistemic_tier":"T3_provisional","created_by_agent_id":null},"outgoing_links":[{"target_artifact_id":"h-SDA-2026-04-26-gap-20260426-001521-01-plasma-nfl-elevation-secondary-to-bbb-associated-873d04c826","link_type":"derives_from","strength":1.0,"evidence":"{\"source\": \"forge_experiment_proposal_generator\", \"method\": \"generate_experiment_proposal\"}"}],"incoming_links":[],"current_artifact_id":"experiment_proposal-afa14449-d6f1-4587-8756-49323c4ea1b5","is_canonical":true,"supersede_chain":["experiment_proposal-afa14449-d6f1-4587-8756-49323c4ea1b5"]}