The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke

Abstract

Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K⁺ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K⁺ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K⁺ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1^-/- mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.

Keywords: KCa3.1; TRAM-34; microglia activation; middle cerebral artery occlusion; potassium channel.

Figure 1.

Scheme illustrating isolation of microglia for confocal microscopy and electrophysiological experiments with CD11b magnetic beads from the infarct area 8 days after MCAO.

Figure 2.

K⁺-channel expression in acutely isolated microglia. (a) Kv1.3 current density increases in microglia from the infarct area after MCAO (28.8 ± 2.0 pA/pF, n = 19) and microglia isolated from the hippocampus following intracerebroventricular LPS injection (22.9 ± 16.6 pA/pF, n = 13) compared to microglia from wild-type control brains (5.0 ± 3.9 pA/pF, n = 16) or microglia from the contralateral side after MCAO (5.7 ± 4.4 pA/pF, n = 18). (b) Example current traces showing Kv1.3's characteristic use-dependence and sensitivity to the Kv1.3 blockers PAP-1 and ShK-L5. (c) Microglia from the contralateral (50.2 ± 35.4 pS/pF, n = 18) and ipsilateral side after MCAO (71.6 ± 34.9 pS/pF, n = 21), as well as microglia isolated from the hippocampus following intracerebroventricular LPS injection (84.0 ± 42.4 pS/pF, n = 13) show higher KCa3.1 current densities than microglia from wild-type control brains (29.7 ± 15.2 pS/pF, n = 16). (d) Example KCa3.1 current traces elicited by a ramp protocol showing the current's sensitive to 1 µM of the KCa3.1-selective blocker TRAM-34. (e) Microglia from both the contralateral side (7.8 ± 5.8 pA/pF, n = 18) and the infarct area (15.1 ± 10.2 pA/pF, n = 21) after MCAO show increased Kir current densities compared to those from wild-type (2.4 ± 2.4 pA/pF, n = 16) or LPS-injected brains (1.9 ± 2.9 pA/pF, n = 13). (f) Representative current traces showing a large Kir current, which was observable in some MCAO microglia, but not in microglia isolated from the hippocampus following intracerebroventricular LPS injection. Data are presented as mean ± S.D. Statistical significance was determined by Student's t-test.

Figure 3.

KCa3.1 and Kv1.3 are expressed on microglia/macrophages in human infarcts. (a) KCa3.1 staining in a 2–3-week-old infarct. KCa3.1 expression is localized to macrophages/microglia (M) and vascular endothelial (E) cells. (b) Fluorescent staining for a microglia/macrophage marker (MAC387) and KCa3.1. (c) Kv1.3 staining in a 14-day old-infarct. (d) Fluorescent staining for a microglia/macrophage marker (MAC387) and Kv1.3. All images are from 5-μm thick paraffin sections.

Figure 4.

KCa3.1 expression on microglia/macrophages in mouse infarcts. (a) Fluorescent staining for KCa3.1 and CD68 in the infarct area of KCa3.1^−/− (a) and wild-type (b) mice five days after MCAO with reperfusion. (c) Higher magnification image showing that KCa3.1 and CD68 do not colocalize. All panels are confocal images from 5-μm thick paraffin sections.

Figure 5.

Genetic KCa3.1 deletion and pharmacological blockade with TRAM-34 reduce infarction and improve neurological deficit. (a) NeuN-defined infarct area in mice subjected to 60 min of MCAO followed by eight days of reperfusion in brain slices 2, 4, 6, and 8 mm from the frontal pole. Shown are vehicle-treated wild-type mice (n = 11), vehicle-treated KCa3.1^−/− mice (n = 17) and wild-type mice treated with TRAM-34 at 40 mg/kg twice daily starting 12 h after reperfusion (n = 11). *p < 0.05, **p < 0.01, ***p < 0.001. (b) Neurological deficit in the 4-score system (normal mouse = 0). (c) Neurological deficit in the 14-score system (normal mouse = 14). *p < 0.05, #p < 0.01, &p < 0.001. All values in panels (a), (b) and (c) are mean ± S.E.M.

Figure 6.

Genetic KCa3.1 deletion and pharmacological blockade with TRAM-34 reduce microglia activation and cytokine production. (a) Microglia/macrophage activation/proliferation/infiltration as determined by the ratio of the Iba-1 positive cells in the ipsi- versus contralateral hemisphere from the 4- and 6-mm slices from all animals in vehicle-treated wild-type mice (n = 11), vehicle-treated KCa3.1^−/− mice (n = 17) and wild-type mice treated with TRAM-34 at 40 mg/kg twice daily starting 12 h after reperfusion (n = 11). (b) Brain cytokine concentrations in the ipsi- and contralateral side eight days after MCAO from vehicle-treated wild-type mice, KCa3.1^−/− mice, TRAM-34-treated wild-type mice and vehicle-treated sham-operated wild-type mice (n = 3 in every case). *p < 0.05, **p < 0.01. Values are mean ± S.E.M.

Figure 7.

TRAM-34 reduces microglia activation and neuronal death in organotypic hippocampal slices. (a) Hippocampal slices were subjected to 60 min of hypoxia and hypoglycemia and stained for Iba-1 (green) to evaluate microglia activation and NeuN (red) to evaluate neurotoxicity 3 days later. TRAM-34 and doxycycline were added 2 h after the end of the hypoxia. (b) Quantification of data shown in (a) from five slices. The number of Iba-1 and NeuN positive cells were counted using ImageJ software and normalized by the total number of DAPI positive cells. (c) Hypoxia induced IL-1β production measured in the medium of the slice culture on day-3 (n = 4). Shown are means ± S.E.M. Statistical analyses were performed using SigmaPlot 11 software (Systat Software, Inc.). Analysis of variance or repeated-measures analysis of variance was used to compare quantitative values from cultures across groups. Tukey's studentized range test was used to adjust for multiple comparisons in post-hoc pairwise tests.