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

BACKGROUND: 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. METHODS: 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. RESULTS: 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+CD45lowLy6clow (microglia) and CD11b+CD45highLy6Clow CNS-MPs but not CD11b+CD45highLy6chigh 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. CONCLUSIONS: We conclude that resident microglia and a subset of CD45highLy6clow 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.

Leptin treatment prevents impaired hypoglycemic counterregulation induced by exposure to severe caloric restriction or exposure to recurrent hypoglycemia.

Hypoglycemia-associated autonomic failure (HAAF) is a maladaptive failure in glucose counterregulation in persons with diabetes (PWD) that is caused by recurrent exposure to hypoglycemia. The adipokine leptin is known to regulate glucose homeostasis, and leptin levels fall following exposure to recurrent hypoglycemia. Yet, little is known regarding how reduced leptin levels influence glucose counterregulation, or if low leptin levels are involved in the development of HAAF. The purpose of this study was to determine the effect of hypoleptinemia on the neuroendocrine responses to hypoglycemia. We utilized two separate experimental paradigms known to induce a hypoleptinemic state: 60% caloric restriction (CR) in mice and three days of recurrent hypoglycemia (3dRH) in rats. A sub-set of animals were also treated with leptin (0.5-1.0 µg/g) during the CR or 3dRH periods. Neuroendocrine responses to hypoglycemia were assessed 60 min following an IP insulin injection on the terminal day of the paradigms. CR mice displayed defects in hypoglycemic counterregulation, indicated by significantly lower glucagon levels relative to controls, 13.5 pmol/L (SD 10.7) versus 64.7 pmol/L (SD 45) (p = 0.002). 3dRH rats displayed reduced epinephrine levels relative to controls, 1900 pg/mL (SD 1052) versus 3670 pg/mL (SD 780) (p = 0.030). Remarkably, leptin treatment during either paradigm completely reversed this effect by normalizing glucagon levels in CR mice, 78.0 pmol/L (SD 47.3) (p = 0.764), and epinephrine levels in 3dRH rats, 2910 pg/mL (SD 1680) (p = 0.522). These findings suggest that hypoleptinemia may be a key signaling event driving the development of HAAF and that leptin treatment may prevent the development of HAAF in PWD.