Temporal profiling of Kv1.3 channel expression in brain mononuclear phagocytes following ischemic stroke

Microglia and CNS-infiltrating monocytes/macrophages (CNS-MPs) perform pro-inflammatory and protective anti-inflammatory functions following ischemic stroke. Selective inhibition of pro-inflammatory responses can be achieved by Kv1.3 channel blockade, resulting in a lower infarct size in the transient middle cerebral artery occlusion (tMCAO) model. Whether beneficial effects of Kv1.3 blockers are mediated by targeting microglia or CNS-infiltrating monocytes/macrophages remains unclear.

In the 30-min tMCAO mouse model, we profiled functional cell-surface Kv1.3 channels and phagocytic properties of acutely isolated CNS-MPs at various timepoints post-reperfusion. Kv1.3 channels were flow cytometrically detected using fluorescein-conjugated Kv1.3-binding peptide ShK-F6CA as well as by immunohistochemistry. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed to measure Kv1.3 (Kcna3) and Kir2.1 (Kcnj2) gene expression. Phagocytosis of 1-μm microspheres by acutely isolated CNS-MPs was measured by flow cytometry.

In flow cytometric assays, Kv1.3 channel expression by CD11b⁺ CNS-MPs was increased between 24 and 72 h post-tMCAO and decreased by 7 days post-tMCAO. Increased Kv1.3 expression was restricted to CD11b⁺CD45^lowLy6c^low (microglia) and CD11b⁺CD45^highLy6C^low CNS-MPs but not CD11b⁺CD45^highLy6c^high inflammatory monocytes/macrophages. In immunohistochemical studies, Kv1.3 protein expression was increased in Iba1⁺ microglia at 24-48 h post-tMCAO. No change in Kv1.3 mRNA in CNS-MPs was observed following tMCAO.

We conclude that resident microglia and a subset of CD45^highLy6c^low CNS-MPs are the likely cellular targets of Kv1.3 blockers and the delayed phase of neuroinflammation is the optimal therapeutic window for Kv1.3 blockade in ischemic stroke.