Role Of K-ATP In The Treatment Of Iptakalim And Memantine During Cerebral Ischemia

Ischemic stroke constitutes a significant human health hazard. It has beenpredicted that, by the year 2020, stroke would become one of the primary causes ofdeath. In the past ten years, clinical trials aiming to figure out suitableneuroprotectants against the debilitating effects of stroke have met with no success.Identifying novel neuroprotectants which can reverse the effects against stroke isbecoming a challenge to both clinicians and scientists. Leading pathogenicmechanisms of ischemic cascade include energy failure, elevation of intracellularCa2+, excitotoxicity, spreading depression, generation of free radicals, BBB disruption,inflammation, and apoptosis. In order to yield sufficient neuroprotection, an idealtherapeutic method for stroke would be the one which rescues multiple brain cells.Therefore, the new targets should be proved to protect the entire neurovascular unit.ATP-sensitive potassium (K-ATP) channels provide a unique link betweencellular energetics and electrical excitability. They are heteromultimers composedof four inwardly rectifier potassium channel pore subunits (Kir6.x) and foursulfonylurea receptors (SURs). These channels are usually closed in normalconditions but are activated rapidly in response to the decreases in intracellularATP/ADP ratio under ischemic conditions. Thus, the opening of K-ATP channelscan get intrinsic protection by regulating membrane potential. Opening K-ATPchannels can hyperpolarize the cell membrane, limiting neuron excitability and Ca2+ influx and thus blocking the subsequent neurotoxic biochemical cascade throughinhibiting activation of N-methyl-D-aspartate receptor-gated ion channel activation.Recent reports have shown that Kir6.2-/- mice displayed enhanced neuronal damageafter ischemic insults and over-expression of Kir6.2 ameliorates the brain ischemicinjury. These findings indicated that K-ATP channels are directly involved inneuroprotection against acute hypoxic or ischemic insult. However, most ofmechanistic studies about the neuroprotection of K-ATP against cerebral ischemiahave been only focused on neurons protection. In fact, accumulating evidences nowshow that glial cells and endothelial cells play an active and important role in thepathophysiology of stroke. Brain energetics, water and ion homeostasis,inflammation, trophic factor production, vascular regulation, neurongenesis, andvasculogenesis are all under the control of glial cells. Endothelial cells form theBrain-blood brarrier (BBB) and participate in inflammatory and immune reactions byproducing solubale mediators. K-ATP channels are widely distributed in brain. Ithas been documented that neurons mainly express Kir6.2-containing K-ATP channelswhereas Kir6.1 is the principal pore-forming subunit of astrocytes and microglia.The recent studies have showed that pericytes also express Kir6.1-based channels.However, it is uncertain that what kind of subunit of K-ATP channel endothelial cellsexpress. Therefore, we proposed that the neuroprotection by K-ATP opening didnot only apply on neurons. However, the exactly mechanisms are still unknown.Therefore, we established the ischemia model by middle cerebral arteryocclusion (MCAO) in both genotypes. In the first part of present study, we used anovel and unselective K-ATP channel opener—iptakalim (IPT) and Kir6.2 knockoutmice to investigated the neuroprotection by opening K-ATP channels. In the secondpart, the neuroprotective effects of NMDA receptor antagonist---memantine againstcerebral ischemia were studied in Kir6.2 knockout mice. Finally, it was illustratedthat K-ATP was an important target of neuroprotectants in stroke. Part I Effects of Kir6.2 knockout on iptakalim-inducedneuroprotectioneuroprotection after cerebral ischemiaAIM: To investigate the effects of Kir6.2 knockout and the opening of K-ATPchannels in ischemia-induced cerebral injury in mice.METHODS: 1)In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. IPT was injected intraperitoneally in a dose of 10mg/kg, 30 minutesbefore middle cerebral artery occlusion (MCAO). Mortality, neurological deficitsand infarct volume were measured in each group 24 hours after ischemia. Doubleimmunofluorescence was used for identifying the subtype of K-ATP channel inendothelial cells and to assess the contents of glia-transmitter D-SerineD-Serine andMMP-9. Immunostaining was taken for observation of endothelial cells, basalmembrane neurons, astrocytes and microglia. Fresh hippocampal homogenateswere used to determine the expressions of connexin43 and MMP-9. The TNF-αproduction was also analyzed by enzyme-linked immunosorbent assay (ELISA). 2)In vitro, primary cortical neuron cultures were prepared from the cortical tissues ofembryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. Cultures were usedafter 7 days in vitro. Oxygen and glucose (OGD) were deprived to induce theneuronal injury. Cortical neurons were incubated with 10μM IPT or 10μM AMPKinhibitor compound c for thirty minutes before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)- -2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis. Western blotting was taken for the analysis of p-AMPKexpression.RESULTS: In vivo,1) Effects of Kir6.2 knockout and IPT pretreatmenton mortality, neurological deficits and infarct volume induced by MCAO Afterischemia, Kir6.2-/- mice exhibited higher mortality while nearly all of Kir6.2+/+ micesurvived. Pretreatment with IPT could increase the survival ratio of Kir6.2-/- mice.Server neurological deficits and larger infarct volume were observed in Kir6.2-/- mice.IPT could attenuate the neurological deficits and decrease the infarct volume in both genotypes. 2) EffectEffects of Kir6.2 knockout and IPT on the structure and functionof neurovasucular unit after ischemia. Endothelial cells expressed Kir6.1 andKir6.2- containing K-ATP channels. Higher production of MMP-9 by endothelialcells was observed in Kir6.2-/- mice three hours after ischemia. Occludin andcollagenIV were then dramatically disrupted in Kir6.2-/- mice. The level of PSD-95expression was significantly decreased. Till 24 hours after ischemia, there was aserver decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group comparedwith that in the Kir6.2+/+ group. Astrocytes displayed reactive changes surroundingthe neurons in CA1 region of Kir6.2+/+ group. The expression of D-SerineD-Serinewas increased while the expression of AQP4 was decreased. However, astrocytesshowed damaged features in CA1 region in Kir6.2-/- mice. The expression ofconnexin43 and D-SerineD-Serine were significantly reduced. AQP4 expressionwas initially dramatically increased and then decreased. Severer microglial wereactivated and much more TNF-αproduction was induced by ischemia in Kir6.2-/-genotype. Pretreatment with IPT inhibited MMP-9 secretion and then prevented thedegradation of occludin and collagenIV and in both genotypes；markedly increasedthe expression of PSD-95 and promoted the neuronal survival in both genotypes;inhibited the activation of astrocytes with the decreased the expression ofD-SerineD-Serine, enhancement the expression of connexin43 and inhibition ofAQP4 expression in Kir6.2+/+ mice; protected astrocytes against ischemia withupregulation of connexin43, D-SerineD-Serine and AQP4 expression in Kir6.2-/- mice;inhibited microglia activation in both genotypes and decreased the production ofTNF-αin Kir6.2-/- genotye;In vitro, Kir6.2 knockout does not altered the level of apoptosis, neuronalsurvival under normal conditions but increased the injury of OGD. IPT increasedthe neuronal survival of two genotypes induced by OGD stimulaiton. After OGDstimulation, the expression of p-AMPK was increased in both genotypes. Higherexpression level of p-AMPK was observed in Kir6.2-/- neurons. IPT inhibited thephosphorylation of AMPK induced by OGD stimulaiton. Inhibition of p-AMPKexpression by compound C could decrease the injury of both genotypes. CONCLUSION:1. Lack of Kir6.2- containing K-ATP channels aggravates endothelial cellsmalfunction, obviously induces neuron apoptosis and astrocyte damage, daramaticallyactivates microglia, which ultimately cause enhancement of cerebral ischemicdamage in Kir6.2-/- mice.2. K-ATP channel opener IPT exerts evident neuroprotective effects in bothgenotype mice through improving the function of entire neurovascular unit.3. All components of neurovascular unit express K-ATP channel. Endothelialcells express Kir6.1- and Kir6.2-containing K-ATP channels. Targeting K-ATPchannels provides a promising therapeutic strategy for protection of neurovascularunit in brain ischemic lesion.Part II Effects of Kir6.2 knockout on memantine-inducedneuroprotection after ischemiaAIM: To investigate whether Kir6.2-containing K-ATP channel participate inmemantine-induced neuroprotection after ischemia and prove that K-ATP is animportant target of neuroprotectants aginast cerebral ischemia.METHODS: In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. Memantine was injected intraperitoneally in a dose of 20mg/kg, 30minutes before MCAO. Since the second day after ischemia, BrdU (100mg, once aday, ip) was administrated to both genotypes followed by 7-day-memantine treatment.Neurological deficits and infarct volume were measured in each group 24 hours afterischemia. Immunostaining was taken for observation of the morphological changesof endothelial cells,basal membrane,neurons, astrocytes, microglia and countingBrdU-positive cells. Immunofluorescence was used to evaluating PSD-95 anddoublecortin (DCX) expression. Primary cortical neuron cultures were preparedfrom the cortical tissues of embryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. In vitro, oxygen and glucose were deprived to induce the neuronal injury.Cortical neurons were incubated with 10μM memantine before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis.RESULTS: 1) Effects of Kir6.2 knockout and memantine pretreatment onneurological deficits and infarct volume induced by MCAO After ischemia,Kir6.2-/- mice exhibited server neurological deficits and larger cerebral infarct volume.Pretreatment with memantine could attenuate the neurological deficits and decreasethe infarct volume but have no impact on Kir6.2-/- mice. 2) Effects of Kir6.2knockout and memantine pretretment on the structure and function ofneurovasucular unit after stroke. Three hours after ischemia, higher productionof MMP-9 by endothelial cells was observed in Kir6.2-/- mice. Occludin andcollagenIV were dramatically disrupted in Kir6.2-/- mice. The expression of PSD-95was much lower in Kir6.2-/- mice. Twanty-four hours after ischemia, there was amuch more decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group.Astrocytes displayed reactive changes in CA1 region of Kir6.2+/+ group. Theexpression of AQP4 was decreased. However, astrocytes showed damaged featuresin Kir6.2-/- mice. AQP4 expression was initially dramatically increased and thendecreased. Severer activation of microglial was observed in Kir6.2-/- genotype. InKir6.2+/+ mice, pretreatment with memantine inhibited MMP-9 secretion and thenprevented the degradation of occludin and collagenIV; prevented the decrease ofPSD-95 in CA1 regions and markedly increased the neuronal survival; inhibited theactivation of astrocytes and microglia; decreased AQP4 expression. In Kir6.2-/- mice,pretreatment with memantine decreased the initial secretion of MMP-9 anddegradation of occludin and collagenIV, but had no effect on the decrease of PSD-95.Till 24 hours after ischemia, the neuronal survival, astrocytes damage and microgliawere not improved. In vitro, the anti-apoptosis effect of memantine on neurons wasabolished by Kir6.2 knockout during OGD stimulation. 3) Effects of Kir6.2knockout and memantine on the neurogenesis after stroke. Daily injection of memantine promoted the proliferation of stem cells located in SVZ in both genotypesafter ischemia. Fourteen days after stroke, all of the Kir6.2-/- mice died.Memantine promoted the migration of stem cells in SVZ of Kir6.2+/+ mice.CONCLUSION: Memantine have no neuroprotective effects on actue cerebralischemic injury of Kir6.2-/- mice. The neuroprotection of memantine in ischemicstroke is dependent on Kir6.2-containing K-ATP channels. The underlyingmechanism is attributed to that memantine can not inhibite the degradation of PSD-95induced by ischemia after Kir6.2 knockout. Memantine could promote neurogenesisafter stroke which will benefit the intrinsic revovery, which is independent onKir6.2-containing K-ATP channels.In summary, our present study has the following new concerns:1. K-ATP channels are promising therapeutic targets for the protection ofneurovascular unit in brain ischemic lesion. Endothelia cells express Kir6.1- andKir6.2- containing K-ATP channels. Kir6.2 knockout damaged the entireneurovascular unit during ischemia. The therapeutic strategy targeted to K-ATP mayoffer a new perspective for the development of new options in ischemic stroketreatment.2. Opening K-ATP channels inhibit the ischemic brain damage throughneurovascular unit protection. Opening K-ATP channels modulate the integrityand function of the neurovascular to play significant neuroprotection in ischemicstroke. The results obtained in present study substantially improve ourunderstanding of K-ATP in stroke.3. Kir6.2 knockout abolishes the neuroprotective effects of memantine.Memantine have no neuroprotective effects on acute cerebral ischemic injury ofKir6.2-/- mice. The underlying mechanism is attributed to that memantine can notinhibite the degradation of PSD-95 induced by ischemia after Kir6.2 knockout.Kir6.2-containing K-ATP channels are involved in the neuroprotection of memantinein cerebral ischemia. Memantine can promote neurogenesis after stroke which willbenefit the intrinsinc revovery. This effect is independent on Kir6.2-containing K-ATP channels.