SAT427 Effects Of Polychlorinated Biphenyls (PCBs) On Transcription Factor NFκB Activation In Neonatal Rat Microglia Primary Culture

Abstract

Abstract Disclosure: C.E. Dressel: None. G.M. Valdez: None. M.R. Bell: None. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are lipophilic and accumulate in the food chain. Despite bans on PCB production, human exposure continues and can cause disruptions in endocrine, nervous, and immune system functions. As such, PCBs may interfere with the actions of microglia, the innate immune cells of the central nervous system. Healthy microglia are critical for neural development, maintenance of neural functions, and responses to stress, injury, or infection. Initial studies of primary neonatal rat microglial cultures indicate that PCBs alter gene expression of proinflammatory cytokine TNF-α and IL-1β in response to a secondary inflammatory lipopolysaccharide (LPS) challenge. Production of these cytokines is, in part, regulated by transcription factor NFκB. This experiment tests the hypothesis that PCBs alter NFκB activation in neonatal microglia. The forebrains of male and female neonatal Sprague-Dawley rats were dissected to remove meninges, homogenized with collagenase and gentle trituration, and plated on coverslips in DMEM FBS+ media to establish a primary mixed glial culture. After one week, microglia were subcultured using mild trypsin treatment and maintained in conditioned media for 24 hours. The microglia were then exposed to either PCB 153 (0.1 µM) or DMSO vehicle control (0.036%) for 18 hours. After 18 hours, the cells were treated with either LPS (0.5 µg/mL) or PBS vehicle control for 4 hours and then fixed. Immunocytochemistry was used to visualize NFκB and identify microglia, with anti-p65 and Ox42 antibodies, respectively. Coverslips were mounted with DAPI-containing media and the cells were imaged via confocal microscopy. In response to immune activation, NFκB typically translocates from the cytosol to the nucleus. Therefore, we assessed NFκB activation by quantifying p65 fluorescent pixel intensity and percentage of area covered in nuclear or cytoplasmic regions according to DAPI and Ox42 channel masks using ImageJ. A two-way ANOVA was used to identify the effects of PCB vs. DMSO exposure and LPS vs. PBS challenge. Preliminary data indicated that LPS increased the ratio of nuclear to cytosolic NFκB, as expected. PCB exposed cells tended to have lower NFκB presence in the nucleus and cell as a whole, but a greater proportion of NFκB staining localized within the nucleus relative to the rest of the cell. With the current sample (n=4-6 per group, 2-3 males and females each), no interactions between PCBs and LPS were observed. Continued work will increase sample size to explore sex differences and the time course of NFκB response to PCBs, which may help broaden our understanding of the mechanisms by which PCBs affect normal brain activity and disrupt healthy endocrine, immune, and neural development. Presentation: Saturday, June 17, 2023