Role Of Kir6.2/K-ATP Channel In Ischemia-reperfusion Induced Injury In C57bL/6J Mice

Stroke, also called cerebrovascular accident, constitutes a significant humanhealth hazard because of its high incidence, morbidity and mortality. In the past tenyears, clinical trials aiming to figure out suitable neuroprotectants against thedebilitating effects of stroke have met with no success. Identifying novelneuroprotectants which can reverse the effects against stroke is becoming a challengeto both clinicians and scientists. Leading pathogenic mechanisms of ischemiccascade include energy failure, elevation of intracellular Ca2+, excitotoxicity, spreadingdepression, generation of free radicals, brain-blood brarrier (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.The recent identification of endogenous neural stem cells (NSCs) and persistentneuronal production in the adult brain suggests a previously unrecognized capacity forself-repair after brain injury. Experimental stroke in adult rodents and primatesincreases neurogenesis in the persistent forebrain subventricular and hippocampaldentate gyrus germinative zones. The adult NSCs are multipotent since they can beinduced to differentiate into neurons, astrocytes and oligodendrocytes, participating inneural repair and functional recovery. How to expand the endogenous progenitorpool, direct migration and differentiation, and promote survivl of the progeny is apriority. Elucidating and then controlling the appropriate regulatory mechanisms in the NSCs will be a major target in the development of regeneration-based therapeuticstrategies for stroke.ATP-sensitive potassium (K-ATP) channel provide a unique link between cellularenergetics and electrical excitability. They are heteromultimers composed of fourinwardly rectifier potassium channel pore subunits (Kir6.x) and four sulfonylureareceptors (SURs). These channels are usually closed in normal brain but areactivated rapidly in response to the decreases in intracellular ATP/ADP ratio underischemic conditions. Ischemic cell death is initiated by changes that result directlyfrom inhibition of oxidative phosphorylation and creat a maelstrom of activity. Thesechanges include decreased ATP, initiation of free radical production by themitochondrial chain, increase cell Na+and membrane depolarization as a result of theloss of ATP substrate for the Na+-K+pump. Opening K-ATP channels canhyperpolarize the cell membrane, limiting neuron excitability and Ca2+influx and thusblocking the subsequent neurotoxic biochemical cascade. Recent reports have shownthat Kir6.2-/-mice displayed enhanced neuronal damage after ischemic insults andover-expression of Kir6.2ameliorates the brain ischemic injury. These findingsindicated that K-ATP channels are directly involved in neuroprotection against acutehypoxic or ischemic insult. However, most of mechanistic studies about theneuroprotection of K-ATP against cerebral ischemia have been only focused on neuronprotection. In fact, accumulating evidences now show that glial cells and endothelialcells play an active and important role in the pathophysiology of stroke. Endothelialcells form the 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 channels(Kir6.2/K-ATP channels) while they are also expressed in astrocytes and microglia.Thus, the widespread tissue distribution of K-ATP channels and their expression ondiscrete cellular populations make them to be the potential target for the novel drugtherapy to protect the brain from ischemic injury. Therefore, we proposed that theneuroprotection by K-ATP opening did not only apply on neurons, but also dependenton the the protection of neurovascular unit.Our lab determined that neural stem cells express K-ATP channels composed of the Kir6.1/SUR1subunits and found that K-ATP channel opener promote adulthippocampal neurogenesis under normal condition and chronic mild stress model.K-ATP channel opener could also promote the subventricular zone (SVZ) andsubgranular zone (SGZ) of hippocampal dentate gyrus (DG) neurogenesis in MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced Parkinson’s disease micemodel. These results suggest that K-ATP channels may contribute to neuronalprotection by promoting neurogenesis and repair. Therefore, these factors highlightthe urgent need for studying the role of Kir6.2-containing K-ATP channels infunctional outcome after focal ischemia. D-Serine (D-Ser), the endogenousco-agonist of N-methyl-D-aspartate (NMDA) receptors, has been recognized as animportant gliotransmitter in the mammalian brain. D-Ser has been implicated inseveral physiological NMDAR-dependent processes, including normal transmission,synaptic plasticity and cell migration in the developing cerebellum. D-Serdysregulation may also play pathological roles in schizophrenia, ageing and acute orchronic neurodegeneration. We have found that K-ATP channel opener couldsignificantly inhibit chronic MPTP treatment induced D-Ser expression enhance in SNastrocytes of mice. It has been documented that MCAO induced a remarkableincrease in the extracellular level of D-Ser during and for four hours after reperfusion.Whether D-Ser is related to increased neurogenesis after ischemic injury and whetherthe D-Ser modulates the functions of NSCs remain unknown.Therefore, the aim of presnt studies is to investigate the role of K-ATP channels instroke. We established the ischemia model by middle cerebral artery occlusion(MCAO) in both wildtype (WT) and Kir6.2knockout (Kir6.2-/-) mice. In the firstpart of present study, we investigated the neuroprotection by opening K-ATP channelsin acute ischemia-reperfusion injury. In the second part, the role of K-ATP channelsin neurogenesis was investageted during neural repair after focal ischemia. In thethird part, the effects of endogenous D-Ser on the proliferation, migration anddifferentiation of NSCs derived from the newborn mouse SVZ were investigated.Finally, it was illustrated that K-ATP was an important target of neuroprotectants instroke and this study will help to reveal the physiological profile of D-Ser further anddevelop a novel therapeuric strategy for the neurological disorders that require nerve cell replenishment, such as neurodegenerative diseases and stroke.Part I Effects of Kir6.2/K-ATP channels on acute phase ofischemia-reperfusion induced injury in C57BL/6J miceAIM: To investigate the effects of Kir6.2/K-ATP channels on acute phase ofischemia-reperfusion induced cerebral injury in mice.METHODS: Focal ischemia were achieved in three-month-old WT and Kir6.2-/-male mice by MCAO for1hour with a modified intralumenal filament technique asdescribed previously. Mortality, neurological deficits and infarct volume weremeasured in each group24hours after ischemia. Changes in vascular permeabilityfollowing MCAO in mice were determined by intravenous injection of2%Evans blue.Immunostaining was taken for observation of endothelial cells, basal membrane,neurons, astrocytes and microglia. Fresh brain homogenates were used to determinethe expressions of heat shock protein70(HSP70), matrix metalloproteinases9(MMP-9), caspase-1, CHOP, glucose-regulated protein78(GRP78) and caspase-12byWestern blotting. The levels of TNF-α and IL-1β were determined by Realtime-PCRand enzyme-linked immunosorbent assay (ELISA).RESULTS:1) MCAO-induced mortality, neurological deficits and infarctvolume were exacerbated in Kir6.2-/-mice.2) Kir6.2knockout increased the BBBdamage after ischemia-reperfusion injury.3) Kir6.2knockout increased theproduction of MMP-9with decreased occludin and collagen IV levels.4) Neuronalinjury in infarct area was exacerbated in Kir6.2-/-mice after MCAO and Kir6.2knockout promoted glial activation in peri-infarct area.5) Kir6.2knockout abolishedMCAO-induced upregulation of HSP70, and enhanced the endoplasmic reticulumstress induced apoptosis protein CHOP, caspase-12expression.6) Kir6.2knockoutpromoted expression of inflammasome protein caspase-1and increased the content ofTNF-α and IL-1β in the brain after MCAO.CONCLUSION: Lacking of Kir6.2/K-ATP channels aggravates endothelial cellsmalfunction, induces neuron apoptosis damage obviously, activates astrocyte andmicroglia dramatically and enhances endoplasmic reticulum stress and inflammatorycytokines release which ultimately cause enhancement of cerebral ischemic damage in Kir6.2-/-mice.Part II The regulatory effects of Kir6.2/K-ATP channel onischemia-induced neurogenesis in C57BL/6J miceAIM: To investigate the role of Kir6.2/K-ATP channel on ischemia-inducedneurogenesis and functional recovery after stroke.METHODS: Focal ischemia was achieved in three-month-old WT and Kir6.2-/-male mice by occlusion of middle cerebral artery (MCAO) with a modifiedintralumenal filament technique as described previously. The ischemia-inducedneurogenesis and the enhanced behavioral recovery on7,14,21,28,35,42and49days were compared between WT and Kir6.2-/-mice. Mice received i.p. injectons offluoxetine (10mg/kg) daily starting on day8after ischemia induction and continuing to35days after MCAO (28days). Neurogenesis was evalutated by counting theBrdU-positive cells and NeuN/BrdU or GFAP/BrdU-positive cells in the SGZ. Nisslstain was used to study the morphology change of neuron and immuohitochemistrywas taken for GFAP and Mac-1. Concentrations of D-Ser in the SVZ andhippocampus were measured at7d,21d and35d after MCAO by high performanceliquid chromatography (HPLC).RESULTS:1) Kir6.2knockout accelerated chronic functional recovery afterMCAO.2) The neurogenesis enhanced in both WT and Kir6.2-/-mice afterischemia-reperfusion injury and chronic fluoxetine treatment increased theneurogenesis in the hippocampus after stroke.3) Kir6.2knockout increased thesurvival of newly born cells in the hippocampus after stroke, but had no influence onthe differentiation of BrdU-positive cells in SGZ.4) Kir6.2knockout abolished thespatial cognitive function injury after MCAO.5) WT mice had significantly moresevere infarct damage and glial activation than Kir6.2-/-mice at7weeks.6) The SVZcontent of D-Ser significantly increased on day7in both genotypic mice and continuedto day21then returned to the baseline on day35. Kir6.2knockout had no effect onthe content of D-Ser after stroke.CONCLUSION: Deficiency of Kir6.2/K-ATP channels reduces infarct damage,promotes functional recovery and BrdU-positive cells survivl7weeks after stroke, indicating K-ATP channels are involved in the modulation of neurorepair afterischemic stroke. The content of D-Ser is corresponding to the increase ofBrdU-positive cells, implying that D-Ser may participate in SVZ neurogenesis afterstroke.Part III The regulatory effects of D-Serine on the functionof mouse neural stem cells in vitroAIM: To investigate the effects and mechanisms of D-Ser on proliferation,migration and differentiation of NSCs in vitro.METHODS: NSCs of newborn mice were isolated and cultured from SVZ tissue.Then we measured the D-Ser levels in culture medium by HPLC analysis.Degradation of endogenous D-Ser with D-amino acid oxidase (DAAO) or blockingNMDA receptor by NMDA receptor antagonist MK-801and addition of exogenousD-Ser, were used to study the regulatory effects of D-Ser on the proliferation,migration and differentiation of NSCs. BrdU incorporation was used to investigatethe proliferation of NSCs. The radial migration of NSCs out of neurospheres wasassayed48hours after plantation. Phenotypic Differentiation of NSCs was determinedby Tuj-1(neuron-specific class III β-tubulin) and GFAP (glial fibrillary acidic protein)double immunofluorescence after7days culture with1%FCS. Intracellularspontaneous calcium oscillations and calcium transients induced by high concentrationKCl were assayed by Fluo-3calcium image. The expressions of GSK3β, ERK1/2and CREB after exhausting the excellular D-Ser were determined by Western blotting.RESULTS:1) The cultured NSCs expressed D-Ser and its biosynthetic enzymeserine racemase (SR).2) The incubation with DAAO exhausting D-Ser couldsignificantly suppress the multiplication rate of NSCs and inhibit the neuronaldifferentiation of NSCs, but it had no influence on NSC migration ability.3)Addition of D-Ser failed to affect the proliferation of NSCs, whereas preincubationwith high concentration of D-Ser (100μM) could alleviate DAAO or MK-801-inducedinhibition of NSC proliferation.4) DAAO exhausting excellular D-Ser couldsignificantly suppress the peak amplitude of glutamate-induced Ca2+transient.5)DAAO inhibited phosphorylation of ERK1/2, CREB and GSK-3β in NSCs. CONCLUSION: NSCs can biosynthesize and release D-Ser that sustains theproliferation and promotes neuronal differentiation of NSCs by acting on NMDAreceptor and regulating [Ca2+]i as well as the ERK1/2-CREB and GSK-3β signalingpathways.In summary, our present study has the following new concerns:1. Kir6.2/K-ATP channels are promising therapeutic targets for theprotection of neurovascular unit in brain ischemic lesion. Kir6.2knockoutdamaged the entire neurovascular unit during ischemia by enhancing endoplasmicreticulum stress response and inflammasome activation. These findings suggest thatK-ATP channels may become new therapeutic targets for proctecting theneurovascular unit.2. Kir6.2/K-ATP channels participate in neural repair afterischemia-reperfusion induced injury. Kir6.2knockout alleviated the chronic phaseischemia-reperfusion injury after MCAO and glial activation, promoted the7-weeksurvival rate of BrdU+cells, decreased ischemia-induced spatial cognitive deficits.The present work provides the evidences that Kir6.2/K-ATP channels are involved inthe stroke recovery.3. Endogenous D-Ser regulates proliferation and neuronal differentiation ofNSCs. The high content of D-Ser in the SVZ after stroke was in accordance with theincrease of BrdU-labelled cells, which suggested that the D-Ser may participate inSVZ neurogenesis afer focal cerebral ischemia. The present work provides directevidences for the first time that NSCs can biosynthesize and release D-Ser that sustainsthe proliferation and promotes neuronal differentiation of NSCs. The therapeuticstrategy target to D-Ser may offer a new perspective for stroke treatment.