Rodríguez, Sónia

Abstract Brain white matter tracts undergo structural and functional changes linked to late-life cognitive decline, but the cellular and molecular contributions to their selective vulnerability are not well defined. In naturally aged mice, we demonstrate that senescent and disease-associated microglia (DAM) phenotypes converge in hippocampus-adjacent white matter. Through gold-standard gene expression and immunolabeling combined with high-dimensional spatial mapping, we identified microglial cell fates in aged white matter characterized by aberrant morphology, microenvironment reorganization, and expression of senescence and DAM markers, including galectin 3 (GAL3/Lgals3), B-cell lymphoma 2 (Bcl2), and cyclin dependent kinase inhibitors, including Cdkn2a/p16ink4a. Pharmacogenetic or pharmacological targeting of p16ink4a or BCL2 reduced white matter GAL3+ DAM abundance and rejuvenated microglial fimbria organization. Our results demonstrate dynamic changes in microglial identity in aged white matter that can be reverted by senotherapeutic intervention to promote homeostatic maintenance in the aged brain.

Abstract Surgical parabiosis is widely used to study the mechanistic influence of the circulating milieu on aging and regeneration. This powerful model presents diverse complications based on age, strain, sex, and other experimental parameters. In young (Y) and old (O) male and female C57BL6 mice, we optimized heterochronic (n=12) and isochronic (n=10 Y-Y, n=7 O-O) parabiosis. Throughout protocol development, we identified several complications including variable responses to anesthesia, external and internal dehiscence, dehydration, and weight loss. We identified and implemented solutions during surgical and post-surgical periods, including titrated anesthesia, reinforced internal sutures, topical agents to promote wound healing, and increased supplementation. By consistently adopting protocol changes we were able to significantly increase survival. Separately, we confirmed the time course of chimerism in heterochronic pairs of C57BL6 and Tg(act-EGFP)Y01Osb (eGFP) mice. Baseline and longitudinal blood samples were collected via tail vein. Flow cytometry was used to visualize GFP-positive cells from the parabiont blood sample. Through blood analysis we found that chimerism occurs as early as 2 days post-operatively. Exploitation of our optimized protocol may enable others to efficiently adopt the surgical parabiosis model to dynamically study mechanisms of aging and regeneration.