Post‐Mortem Delay Induces Distinct Transcriptional and Morphological Changes in Microglia With Age‐Specific Patterns

Postmortem tissue is a vital resource for transcriptomic studies of human microglia, yet the influence of postmortem delay (PMD) on microglial states, particularly in aging, remains insufficiently understood. Here, we examined the impact of PMD in young and aged male mice, with a particular focus on aging-associated primed microglia. We performed bulk RNA sequencing on Dectin-1-high and -low microglia isolated after PMDs of 0, 6, or 12 h, with Dectin-1 serving as a marker of primed microglia. PMD did not obscure aging-associated signatures or reduce viability, but consistently altered gene expression profiles. Upregulated pathways included mitochondrial, heat-shock, and apoptosis regulation responses, while actin cytoskeleton regulation was downregulated. These effects differed between young and aged animals, and between primed and non-primed microglia, with attenuation in primed subsets. Reanalysis of human single-cell and single-nucleus datasets confirmed that PMD-associated signatures identified in our dataset, particularly those in aging-related Dectin-1low microglia, correlate with PMD in human datasets and display similar enrichment patterns. Morphological analysis in fixed brain tissue from the same animals revealed that postmortem delay reduced the cell shape complexity of cortical microglia in young mice, mimicking morphological changes in the aged brain. In contrast, the morphology of aged microglia remained unchanged by postmortem delay. Taken together, these findings suggest that postmortem delay introduces subtle yet consistent transcriptional and morphological changes in microglia that can confound the interpretation of aging- and disease-related phenotypes. These results highlight the importance of controlling for postmortem effects in studies using human postmortem tissue.