Expression Of Kv1.2 In Microglia And Its Putative Roles In Modulating Production Of Proinflammatory Cytokines And Reactive Oxygen Species

[Abstract]Objective:Microglial cells monitor the state of health of the surrounding tissues in the central nervous system and act as a neuropathology sensor.They are responsive to various stimuli including infections and ischemia and are activated in some dieases.Microglial cells are endowed with different potassium ion channels but their expression and involvement in specific functions has remained to be fully explored.In this connection,ion channels that modulate the membrane potential control the electrophysiological characteristic of microglia.A fuller understanding of the ion channels is therefore vital in elucidating the mechanism of microglial activation.Method:Immunohistochemistry for Kv1.2 was carried out using the ABC method.Double immunofluorescence labeling for Kv1.2 and OX42/Lectin(The mark of microglia)was used to determine the Kv1.2 cellular location.P1 postnatal rats were kept in a chamber filled with a gas mixture of 5%oxygen and 95%nitrogen for 3 hours.Quantitative RT-PCR was carried out on a Light Cycler 3 instrument to analysis the IL-1βand TNFαmRNA expression.Intracellular levels of potassium were assessed by determination of PBFI fluorescence.In order to confirm that the changes in intracellular potassium concentration and release of cytokines by microglial cells under different conditions involved Kv1.2,TsTx,a specific and a potent Kv1.2 current blocker were applied to the cells in culture.Results:This study has shown Kv1.2 expression in the amoeboid microglia in the postnatal rats' brain between P1 and P10 days.At P14,Kv1.2 expression was localized in the ramified microglia and at P21,it was hardly detected.In P1 rats exposed to hypoxia,Kv1.2 immunoreactivity in amoeboid microglia was markedly enhanced.RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cell culture and murine BV-2 cells. Furthermore,it was up-regulated when the cells were subjected to hypoxia. Additionally,Kv1.2 mRNA expression coupled by that of IL-1βand TNF-αincreased following ATP,and LPS application.Concomitantly,the intracellular potassium concentration in activated microglia as determined by PBFI fluorescence decreased. Blockade of Kv1.2 channel with TsTx resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1βand TNF-αas well as production of intracellular ROS.Conclusion:Kv1.2 expression change is related to the specific necessary of microglia during rat developing.Its expression increase related to the microglia activation.We conclude that Kv1.2 in microglia is sensitive to hypoxia,ATP and LPS.Its up-regulated expression as elicited by the external stimuli may be linked to IL-1βand TNFαexpression and ROS production. Hence,optimizing the dosage of TsTx in suppression of Kv1.2 function may be a potential therapeutic means in ameliorating neuroinflammation in which microglial activation is implicated.