Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K+ channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ∼50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1+-activated microglia and TUNEL-positive neurons as well as increases in NeuN+ neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.
AspeyBSTaylorFLTerruliMHarrisonMJ (2000) Temporary middle cerebral artery occlusion in the rat: consistent protocol for a model of stroke and reperfusion. Neuropathol Appl Neurobiol26:232–42
2.
AtagaKISmithWRDe CastroLMSwerdlowPSaunthararajahYCastroOVichinskyEKutlarAOrringerEPRigdonGCStockerJW (2008) Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia. Blood111:3991–7
3.
AtagaKIStockerJ (2009) Senicapoc (ICA-17043): a potential therapy for the prevention and treatment of hemolysis-associated complications in sickle cell anemia. Expert Opin Investig Drugs18:231–9
4.
BegenisichTNakamotoTOvittCENehrkeKBrugnaraCAlperSLMelvinJE (2004) Physiological roles of the intermediate conductance, Ca2+-activated potassium channel Kcnn4. J Biol Chem279:47681–7
5.
BeschornerRSchluesenerHJGozalanFMeyermannRSchwabJM (2002) Infiltrating CD14+ monocytes and expression of CD14 by activated parenchymal microglia/macrophages contribute to the pool of CD14+ cells in ischemic brain lesions. J Neuroimmunol126:107–15
6.
BrahlerSKaisthaASchmidtVJWolfleSEBuschCKaisthaBPKacikMHasenauALGrgicISiHBondCTAdelmanJPWulffHde WitCHoyerJKohlerR (2009) Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and causes hypertension. Circulation119:2323–32
7.
BusseREdwardsGFeletouMFlemingIVanhouttePMWestonAH (2002) EDHF: bringing the concepts together. Trends Pharmacol Sci23:374–80
8.
CampanellaMScioratiCTarozzoGBeltramoM (2002) Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke33:586–92
9.
CipollaMJGodfreyJA (2010) Effect of hyperglycemia on brain penetrating arterioles and cerebral blood flow before and after ischemia/reperfusion. Transl Stroke Res1:127–34
10.
DamoiseauxJGDoppEACalameWChaoDMacPhersonGGDijkstraCD (1994) Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology83:140–7
11.
De RyckMVan ReemptsJBorgersMWauquierAJanssenPA (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke20:1383–90
12.
del ZoppoGGinisIHallenbeckJMIadecolaCWangXFeuersteinGZ (2000) Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol10:95–112
13.
DenesAVidyasagarRFengJNarvainenJMcCollBWKauppinenRAAllanSM (2007) Proliferating resident microglia after focal cerebral ischaemia in mice. J Cereb Blood Flow Metab27:1941–53
14.
DittmarMSVatankhahBFehmNPSchuiererGBogdahnUHornMSchlachetzkiF (2006) Fischer-344 rats are unsuitable for the MCAO filament model due to their cerebrovascular anatomy. J Neurosci Methods156:50–4
15.
EmsleyHCSmithCJGeorgiouRFVailAHopkinsSJRothwellNJTyrrellPJ (2005) A randomised phase II study of interleukin-1 receptor antagonist in acute stroke patients. J Neurol Neurosurg Psychiatry76: 1366–72
16.
GhanshaniSWulffHMillerMJRohmHNebenAGutmanGACahalanMDChandyKG (2000) Up-regulation of the IKCa1 potassium channel during T-cell activation: molecular mechanism and functional consequences. J Biol Chem275:37137–49
17.
HanleyPJMussetBReniguntaVLimbergSHDalpkeAHSusRHeegKMPreisig-MullerRDautJ (2004) Extracellular ATP induces oscillations of intracellular Ca2+ and membrane potential and promotes transcription of IL-6 in macrophages. Proc Natl Acad Sci USA101:9479–84
18.
KaushalVKoeberlePDWangYSchlichterLC (2007) The Ca2+-activated K+ channel KCNN4/KCa3.1 contributes to microglia activation and nitric oxide-dependent neurodegeneration. J Neurosci27:234–44
19.
KhannaRRoyLZhuXSchlichterLC (2001) K+ channels and the microglial respiratory burst. Am J Physiol Cell Physiol280:C796–806
20.
KohlerRWulffHEichlerIKneifelMNeumannDKnorrAGrgicIKampfeDSiHWibawaJRealRBornerKBrakemeierSOrzechowskiHDReuschHPPaulMChandyKGHoyerJ (2003) Blockade of the intermediate-conductance calcium-activated potassium channel as a new therapeutic strategy for restenosis. Circulation108:1119–25
21.
KurianMMBerwickZCTuneJD (2011) Contribution of IKCa channels to the control of coronary blood flow. Exp Biol Med (Maywood)236:621–7
22.
LamplYBoazMGiladRLorberboymMDabbyRRapoportAAnca-HershkowitzMSadehM (2007) Minocycline treatment in acute stroke: an open-label, evaluator-blinded study. Neurology69:1404–10
23.
LehrHAMankoffDACorwinDSanteusanioGGownAM (1997) Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. JHistochem Cytochem45:1559–65
24.
LieszASuri-PayerEVeltkampCDoerrHSommerCRivestSGieseTVeltkampR (2009) Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med15:192–9
25.
LongaEZWeinsteinPRCarlsonSCumminsR (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke20:84–91
26.
MaezawaIZiminPWulffHJinLW (2010) A-beta oligomer at low nanomolar concentrations activates 2374 microglia and induces microglial neurotoxicity. J Biol Chem286:3693–706
27.
MarrelliSPKhorovetsAJohnsonTDChildresWFBryanRMJr. (1999) P2 purinoceptor-mediated dilations in the rat middle cerebral artery after ischemia-reperfusion. Am J Physiol276:H33–41
28.
MaulerFHinzVHorvathESchuhmacherJHofmannHAWirtzSHahnMGUrbahnsK (2004) Selective intermediate-/small-conductance calcium-activated potassium channel (KCNN4) blockers are potent and effective therapeutics in experimental brain oedema and traumatic brain injury caused by acute subdural haematoma. Eur J Neurosci20:1761–8
29.
McNeishAJSandowSLNeylonCBChenMXDoraKAGarlandCJ (2006) Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery. Stroke37:1277–82
30.
MenziesSAHoffJTBetzAL (1992) Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model. Neurosurgery31:100–7
31.
O'DonnellMETranLLamTILiuXBAndersonSE (2004) Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab24:1046–56
32.
OffnerHSubramanianSParkerSMAfentoulisMEVandenbarkAAHurnPD (2006) Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab26:654–65
33.
PenaTLChenSHKoniecznySFRaneSG (2000) Ras/MEK/ERK Up-regulation of the fibroblast KCa channel FIK is a common mechanism for basic fibroblast growth factor and transforming growth factor-beta suppression of myogenesis. J Biol Chem275:13677–82
34.
PriceCJWangDMenonDKGuadagnoJVCleijMFryerTAigbirhioFBaronJCWarburtonEA (2006) Intrinsic activated microglia map to the peri-infarct zone in the subacute phase of ischemic stroke. Stroke37:1749–53
35.
ReichEPCuiLYangLPugliese-SivoCGolovkoAPetroMVassilevaGChuINomeirAAZhangLKLiangXKozlowskiJANarulaSKZavodnyPJChouCC (2005) Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice. Eur J Immunol35:1027–36
36.
SchillingMBesselmannMLeonhardCMuellerMRingelsteinEBKieferR (2003) Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol183:25–33
37.
SchillingTStockCSchwabAEderC (2004) Functional importance of Ca2+-activated K+ channels for lysopho-sphatidic acid-induced microglia migration. Eur J Neurosci19:1469–74
38.
SchlattmannPDirnaglU (2010) Statistics in experimental cerebrovascular research: comparison of more than two groups with a continuous outcome variable. J Cereb Blood Flow Metab30:1558–63
39.
ShichitaTSugiyamaYOoboshiHSugimoriHNakagawaRTakadaIIwakiTOkadaYIidaMCuaDJIwakuraYYoshimuraA (2009) Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nat Med15:946–50
40.
SiHHeykenWTWolfleSETysiacMSchubertRGrgicIVilianovichLGiebingGMaierTGrossVBaderMde WitCHoyerJKohlerR (2006) Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel. Circ Res99:537–44
41.
ToyamaKWulffHChandyKGAzamPRamanGSaitoTFujiwaraYMattsonDLDasSMelvinJEPrattPFHatoumOAGuttermanDDHarderDRMiuraH (2008) The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans. J Clin Invest118:3025–37
42.
WeinsteinJRKoernerIPMollerT (2010) Microglia in ischemic brain injury. Future Neurol5:227–46
43.
WulffHCastleNA (2010) Therapeutic potential of KCa3.1 blockers: recent advances and promising trends. Expert Rev Clin Pharmacol3:385–96
44.
WulffHKnausHGPenningtonMChandyKG (2004) K+ channel expression during B-cell differentiation: implications for immunomodulation and autoimmunity. J Immunol173:776–86
45.
WulffHMillerMJHaenselWGrissmerSCahalanMDChandyKG (2000) Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci USA97:8151–6
46.
YenariMAKauppinenTMSwansonRA (2010) Microglial activation in stroke: therapeutic targets. Neurotherapeutics7:378–91
47.
YrjanheikkiJKeinanenRPellikkaMHokfeltTKoistinahoJ (1998) Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA95:15769–74
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