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Characterizing the Effect of Clodronate in the Locus Coeruleus During Social Stress

Social stress is a leading cause of psychiatric disorders such as depression and anxiety. Studies have linked social stress‐evoked neuroinflammation to the development of psychiatric disorders, in which elevated pro‐inflammatory cytokines are observed in both plasma and brain tissue of individuals diagnosed with depression. Our goal was to study how microglia, the resident immune cells of the brain, respond to social stress within the stress sensitive locus coeruleus (LC), by measuring the inflammatory‐regulating Chemokine Ligand 1 (CX3CL1), High Mobility Group Box 1 (HMGB1), and microglia‐specific ionized calcium‐binding adapter molecule 1 (IBA1). This study identified a method capable of inducing brain region specific reduction of microglial cells in the LC by administering mannosylated liposomal clodronate (Encapsula NanoSciences). We determined that 25 μg of clodronate (CLD) administered directly into the LC via the mannosylated lipid‐based nanoparticle was selectively toxic to microglia of the LC. Pilot studies also determined that 25 ug of the mannosylated liposomal CLD in the central amygdala reduced the population of microglia in that region by approximately 50%. With this knowledge we sought to determine the effects of CLD within the LC at different timepoints during repeated social stress using a witness stress (WS) model in which females were forced daily to witness social defeat between two males. The two experiments conducted in this study included female rats with a bilateral cannula implanted into the LC. In the first study, rats were administered either Veh (empty liposome) or CLD (25ug/side), three days later all rats were exposed to stress on two consecutive days and tissue and blood collected 1hr post stress. In the second experiment, rats were divided into four stress/treatment groups: Con+Veh, Con+CLD, WS+Veh, and WS+CLD. 3 days post treatment, rats were subjected to a handled control condition or WS (5 days, 15 min/day). 5 days after the final stress/control, rats were sacrificed and tissue from the LC was collected and homogenized to conduct immune assessments of protein levels within this region. Western blotting assays were performedto assess levels for CX3CL1, HMGB1, and IBA1. Our findings from study 1 indicate that upon the second exposure to stress, CX3CL1 levels were not different between vehicle and CLD groups, indicating that neuronal silencing of microglia had not been recruited or inhibited. As would be expected, HMGB1 expression was reduced in the CLD treated group compared with vehicle, suggesting that neuroinflammatory priming was delayed by the administration of CLD. Moreover, in study 2, chronic social stress produced an observable increase in CX3CL1 and a decrease in HMGB1 levels in the LC of WS female rats, regardless of treatment. Moreover, by 5 days after the last stress/control there was an observed increase in Iba1 levels, indicating repopulation of the microglia. These data suggest after long‐term stress there is a potential delayed repopulation of microglia within the LC, accompanied with a primed inhibitory response from neurons within this region that potentially prevent an exacerbated stress response from these new cells.