A Novel 3D Imaging Pipeline for Analyzing Efficacy of Compounds on Amyloid-Beta Plaque Dynamics in Pre-clinical Alzheimer’s Disease Animal Models

Significance of the Topic

Amyloid-beta (Aβ) plaque accumulation is a central hallmark of Alzheimer’s disease (AD) pathology and a primary target for therapeutic development. Conventional assays for Aβ rely on homogenized tissue extracts or small region analyses, limiting spatial resolution and preventing secondary investigations. A high-throughput, whole-organ imaging approach that preserves anatomical context and permits follow-up molecular studies could greatly advance preclinical AD research and drug screening.

Objectives and Overview of the Study

• Develop and validate a novel Serial Two-Photon Plus (STP2) imaging pipeline for quantitative, region-specific mapping of Aβ plaques in mouse brains.
• Integrate high-resolution 3D volumetric data with secondary proteomic imaging (MALDI HiPLEX-IHC) to correlate plaque burden with molecular signatures.
• Demonstrate the temporal progression of Aβ deposition in two transgenic AD mouse models (5XFAD and SAA) across multiple ages.

Methodology and Instrumentation

• Animal Models: Female 5XFAD and SAA mice received intraperitoneal injection of methoxy-X04 (0.5 mg/kg) to label Aβ plaques, followed by perfusion and fixation.
• STP2 Imaging: Intact brains embedded in agarose were sectioned at 50–100 µm and imaged using a TissueVision TissueCyte Serial Two-Photon Plus system (780 nm excitation). Three fluorescence channels captured tissue autofluorescence, vasculature (DyLight-594 lectin), microglia (IBA-1), and methoxy-X04 signal.
• Atlas Registration: Each 3D dataset was aligned to the Allen Mouse Common Coordinate Framework v3 using custom image-analysis algorithms to assign plaque density to defined anatomical regions.
• Secondary Proteomic Imaging: Selected sections underwent multiplexed MALDI HiPLEX-IHC (AmberGen) and mass spectrometry imaging (Bruker Daltonics) to detect 14 protein targets (e.g., Aβ42, pTau, GLUT-1, myelin, neurogranin, SNCA, synapsin), which were co-registered with STP2 volumes.

Main Results and Discussion

• 3D Plaque Mapping: STP2 imaging revealed region-specific plaque accumulation over time, with notable increases in hippocampal formation, thalamus, and cortical subplate between 2 and 6 months of age in 5XFAD mice.
• Quantitative Metrics: Fractional plaque area was computed for each atlas region, enabling statistical comparison (*p < 0.05) and temporal profiling of disease progression.
• Proteomic Correlation: MALDI HiPLEX-IHC data aligned with STP2 volumes revealed spatial colocalization of Aβ plaques with changes in vascular lectin labeling and microglial IBA-1 signal, supporting analysis of cerebral amyloid angiopathy.
• Multiplex Signatures: Ion images of GLUT-1, SNCA, pTau, and myelin provided molecular context to structural pathology, illustrating the potential to track proteomic alterations in targeted brain regions.

Benefits and Practical Applications of the Method

• High Throughput: Whole-brain STP2 imaging accelerates quantitative mapping of Aβ burden without destructive homogenization.
• Anatomical Precision: Atlas-based registration yields region-specific metrics, supporting detailed phenotypic comparisons in preclinical models.
• Multimodal Integration: Combining volumetric fluorescence imaging with secondary proteomic mass spectrometry enriches mechanistic insights and biomarker discovery.
• Translational Utility: The platform generates comprehensive spatial-temporal data suitable for drug efficacy studies and cross-model comparisons.

Future Trends and Potential Applications

• Expansion to Other Pathologies: Application of STP2 plus MALDI HiPLEX-IHC to tauopathies, synucleinopathies, and vascular dementia models.
• Higher Plex Imaging: Development of larger antibody panels and novel mass tags to profile broader proteomic or metabolomic landscapes.
• Automated Analytics: Incorporation of machine learning algorithms for segmentation, plaque classification, and multimodal pattern recognition.
• Human Tissue Translation: Adaptation of the pipeline for cleared post-mortem human brain specimens to bridge preclinical and clinical research.

Conclusion

The Serial Two-Photon Plus pipeline, augmented by atlas registration and MALDI HiPLEX-IHC proteomic imaging, provides a powerful platform for high-resolution, region-specific quantitation of Aβ pathology in mouse models of Alzheimer’s disease. By enabling comprehensive spatial and molecular analysis, this approach enhances the fidelity of preclinical studies and supports the identification of novel therapeutic targets.

Reference

• Ferron G., Knesis A., Linehan S. et al. A Novel 3D Imaging Pipeline for Analyzing Efficacy of Compounds on Amyloid-Beta Plaque. Poster ThP 552, Society for Neuroscience Abstract 312475.