Advanced quantitative mapping of Alzheimer’s disease neuropathology and microglial activation in post-mortem hippocampal tissue

Abstract We developed a high-throughput imaging workflow to spatially map Alzheimer’s disease (AD) pathology in postmortem hippocampal and medial temporal lobe sections from 65 University of Southern California Alzheimer's Disease Research Center (USC ADRC) cases classified by low, intermediate and high levels of AD neuropathologic change (ADNC). Sections were stained for microglia (Iba1), amyloid-β (4G8), and neurofibrillary tangles (NFTs; Gallyas), and analyzed using pixel-based machine learning (Ilastik). Amyloid pathology was classified as dense, diffuse, or intracellular amyloid precursor protein (APP)-positive; microglia were categorized into ramified, rod-like, and amoeboid morphologies. Diffuse amyloid plaques increased most significantly across disease, particularly in the subiculum and dentate gyrus molecular layer, while dense plaques and intracellular APP were concentrated in entorhinal and perirhinal cortices. NFT burden was elevated in males, especially in parahippocampal cortical areas. Microglial morphology shifted with AD progression, showing reduced ramified and increased amoeboid profiles in high ADNC cases. Rod microglia in CA regions were correlated with amyloid in high ADNC and tau in low ADNC cases. Memory impairment correlated more strongly with amyloid pathology in apolipoprotein E (ApoE) ε4 non-carriers, with females showing greater amyloid burden and males more NFT-related decline. These findings reveal region- and sex-specific patterns of pathology and neuroinflammation, offering new insights into mechanisms driving cognitive decline and informing future diagnostic and therapeutic strategies.