Methadone Requires the Co-Activation of µ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells.

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates µ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express µ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of µ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between µ-opioid and TLR4 receptors.

Transcranial Focal Electric Stimulation Avoids P-Glycoprotein Over-Expression during Electrical Amygdala Kindling and Delays Epileptogenesis in Rats.

Recent evidence suggests that P-glycoprotein (P-gp) overexpression mediates hyperexcitability and is associated with epileptogenesis. Transcranial focal electrical stimulation (TFS) delays epileptogenesis and inhibits P-gp overexpression after a generalized seizure. Here, first we measured P-gp expression during epileptogenesis and second, we assessed if TFS antiepileptogenic effect was related with P-gp overexpression avoidance. Male Wistar rats were implanted in right basolateral amygdala and stimulated daily for electrical amygdala kindling (EAK), P-gp expression was assessed during epileptogenesis in relevant brain areas. Stage I group showed 85% increase in P-gp in ipsilateral hippocampus (p < 0.001). Stage III group presented 58% and 57% increase in P-gp in both hippocampi (p < 0.05). Kindled group had 92% and 90% increase in P-gp in both hippocampi (p < 0.01), and 93% and 143% increase in both neocortices (p < 0.01). For the second experiment, TFS was administrated daily after each EAK stimulation for 20 days and P-gp concentration was assessed. No changes were found in the TFS group (p > 0.05). Kindled group showed 132% and 138% increase in P-gp in both hippocampi (p < 0.001) and 51% and 92% increase in both cortices (p < 0.001). Kindled + TFS group presented no changes (p > 0.05). Our experiments revealed that progression of EAK is associated with increased P-gp expression. These changes are structure-specific and dependent on seizure severity. EAK-induced P-gp overexpression would be associated with neuronal hyperexcitability and thus, epileptogenesis. P-gp could be a novel therapeutical target to avoid epileptogenesis. In accordance with this, TFS inhibited P-gp overexpression and interfered with EAK. An important limitation of the present study is that P-gp neuronal expression was not evaluated under the different experimental conditions. Future studies should be carried out to determine P-gp neuronal overexpression in hyperexcitable networks during epileptogenesis. The TFS-induced lessening of P-gp overexpression could be a novel therapeutical strategy to avoid epileptogenesis in high-risk patients.