(251) Opioids and microglia: a murine-derived microglia stable cell line study

Abstract

The development of opioid hyperalgesia following long-term opioid use is a critical impediment in treating chronic pain. Activated microglia has been identified as a critical mediator in the development of opioid hyperalgesia. The mechanism by which opioids activate microglia remains unknown. To explore this question, we used a stable cell line of mouse-derived microglial cells, which allowed us to isolate the direct effect of opioids on microglia in the absence of neurons. The BV-2 microglial cell line, derived from mouse microglial cells, was cultured in vitro. Cells were exposed to 10uM morphine for 48 hours and the degree of microglial activation was assayed with immunohistochemistry using an antibody against IBA-1 and western blot measuring BDNF expression. Standard qPCR was run to probe the expression of BDNF and opioid receptor mRNA in this cell line before and after exposure to opioids. Glial inhibitors (+) naloxone and minocycline were co-administered with morphine and the effect on microglial activation and BDNF expression was measured. BV-2 cells cultured with morphine for 48 hours caused significant increase in BDNF expression. This was blocked by co-treatment with minocycline or (+) naloxone, the opioid inactive enantiomer of naloxone. Kappa and mu opioid mRNA was detected in resting BV-2 cells, with mu opioid receptor expression increasing after morphine treatment. This study found that microglia express opioid receptors and suggest opioids are able to directly activate microglia in vitro. (+) naloxone blocked microglial activation, suggesting (+) naloxone inhibits microglial activation via a non-opioid target. Studies elucidating the molecular target of (+) naloxone in this cell line are ongoing. The development of opioid hyperalgesia following long-term opioid use is a critical impediment in treating chronic pain. Activated microglia has been identified as a critical mediator in the development of opioid hyperalgesia. The mechanism by which opioids activate microglia remains unknown. To explore this question, we used a stable cell line of mouse-derived microglial cells, which allowed us to isolate the direct effect of opioids on microglia in the absence of neurons. The BV-2 microglial cell line, derived from mouse microglial cells, was cultured in vitro. Cells were exposed to 10uM morphine for 48 hours and the degree of microglial activation was assayed with immunohistochemistry using an antibody against IBA-1 and western blot measuring BDNF expression. Standard qPCR was run to probe the expression of BDNF and opioid receptor mRNA in this cell line before and after exposure to opioids. Glial inhibitors (+) naloxone and minocycline were co-administered with morphine and the effect on microglial activation and BDNF expression was measured. BV-2 cells cultured with morphine for 48 hours caused significant increase in BDNF expression. This was blocked by co-treatment with minocycline or (+) naloxone, the opioid inactive enantiomer of naloxone. Kappa and mu opioid mRNA was detected in resting BV-2 cells, with mu opioid receptor expression increasing after morphine treatment. This study found that microglia express opioid receptors and suggest opioids are able to directly activate microglia in vitro. (+) naloxone blocked microglial activation, suggesting (+) naloxone inhibits microglial activation via a non-opioid target. Studies elucidating the molecular target of (+) naloxone in this cell line are ongoing.