The Role Of Aquaporin-4 In Ischemia/reperfusion Induced Injury

Stroke,a disorder encompassing all cerebrovascular accidents,is a public health problem of immense proportions across the globe.As the second leading cause of death behind ischemic heart disease,it leads to serious long-term disability in adults.About 80%of strokes are caused by focal cerebral artery occlusion(acute ischemic stroke,AIS)and brain injury is thought to result from a cascade of events from energy depletion to cell death.Many efforts are directed in the management of AIS but few have proved effective.Numerous pharmaceutical agents targeting one or more cell death pathways showed no improvement in patients' prognosis for not "stopping the stroke clock".These factors highlight the urgent need for new therapeutic options for AIS treatment.Over the past years,the knowledge of cerebral hypersensitivity to ischemia is the main reason for nihilism surrounding the development of remedies for AIS.Substantial clinical trials of thrombolytic and neuroprotecants have been conducted over the last two decades. Neurologists have searched for a long time to find a potential agent among N-methyl-D-aspartate(NMDA)antagonists,radical scavengers, antiinflammatory agents,calcium antagonists,sodium or potassium channel blockers,caspases inhibitors,and neurotrophic factors,but with no success so far.Initial attempts at translating the positive effects of these agents from animal models to patients have either failed.Current experimental support reveals that glia or endotheliocytes were suffered as well in AIS,suggesting the impairment of neurovascular unit may probably be the key point.As an integral unit serving multiple and diverse roles,rescue of neurovascular unit integrity has been considered as a promising strategy for AIS therapeutics.It is believed that astrocytes are critical determinants in neurovascular unit since besides coupling to neuron,they link vascular signal to brain.These glial cells participate in actual information processing and neuronal control of vascular tone by releasing gliotransmitters or vasoactive molecules.They are critically affected in, and contribute to,the anoxic/ischemic process and changes in neurone-astrocyte-endothelial cell signalling pathway may contribute mostly to ischemia reperfusion(IR)injury to central nervous system (CNS)elements.Owing to the pivotal role of astrocytes in neurovascular unit,agents targeting astrocytes regulation open new perspectives for AIS management.In the CNS,aquaporin-4(AQP4)is the predominant isoform which is localized on ependymal cells lining the ventricles and astrocytes membrane.Its expression in neurovascular unit is restricted to the perivascular glial processes,suggesting AQP4 involvement in the maintenance of cerebral homeostasis and may be the key point in astrocytes modulation to control diverse physiological processes. Accumulating data have shown that AQP4 depletion or down-regulation caused astrocytes dysfunction such as reduced membrane water permeability,impaired cell growth or migration,attenuated potassium buffering,and altered cytoskeleton rearrangement.It also serves as a component in microglia activation or blood-brain barrier(BBB)integrity with experimental support.By far,AQP4 has gained sufficient attraction in numerous cerebral disorders including edema,traumatic injury,tumor,infection,and epilepsy,whereas little is known about its contribution to AIS.Due to most studies are still dwelling on AQP4 as a water transporter,the illumination of its significance in neurovascular unit may offer prospective clinical therapeutic benefits. In the present study,we investigate the role of AQP4 in the proceeding of IR in vivo and in vitro by using AQP4 gene knockout mice. The results revealed here will improve our understanding on the neurobiology of AQP4 and provide a new target for developing therapeutic options for AIS management.Partâ… Effects of AQP4 deficiency on the brain injury induced by IRAIM:To investigate the role of AQP4 in IR and the impact of AQP4 knockout on the protective actions of fluoxetine(Flu).METHODS:Focal IR was achieved in three-month-old wildtype (AQP4^+/+)and AQP4 knockout(AQP4^-/-)male mice by transient occlusion of middle cerebral artery(tMCAO)with a modified intralumenal filament technique as described previously.Mortality, neurological deficits and infarct volume were measured in each group. Immunostaining was taken for NeuN,GFAP and MAC1 expression. Brain water content and AQP4 level were also analyzed.RESULTS:Compared with wildtype mice,1)AQP4^-/-genotype exhibited larger increases in mortality,neurological deficits and infarct volume;The dependence on the duration of ischemia was cancelled by AQP4 deficiency;2)Severer loss of CA1 neurons in hippocampus and inhibited astroglial proliferation or microglial activation induced by IR were observed in AQP4^-/-genotype;3)Pool of AQP4 was down-regulated at 24 h and up-regulated at 72 h after tMCAO,exhibiting a time-course change;4)Brain water content was higher in ipsilateral hemisphere in AQP4^-/-mice.After administration of Flu(40 mg/kg,i.p.),5)Mortality, neurological deficits and infarct volume of AQP4^+/+genotype were decreased;6)The loss of CA1 neurons and astroglial proliferation or microglial activation in the core were alleviated;7)Up- and down-regulation of AQP4(at 24 or 72 h,respectively)were detected;8) Brain water content was decreased strikingly at 72 h in AQP4^+/+mice. All these effects of Flu were abolished in AQP4^-/-genotype.CONCLUSION:AQP4 deficiency aggravates IR-induced injury, and the neuroprotection of Flu might be due to the regulation of AQP4 expression.Partâ…¡Effects of AQP4 deficiency on glutamate-induced excitotoxicity1.Effects of AQP4 deficiency on glutamate-induced excitotoxicity in astrocyte-neuron co-culturesAIM:To investigate the effects of AQP4 knockout on glutamate (Glu)induced excitotoxicity.METHODS:Primary astrocyte and neuron cultures were prepared from wildtype and homozygous mutant mice.The co-culture system was achieved by inverting the coverslips bearing astrocytes and all cells were exposed to Glu(100μM).Immunocytochemistry for GFAP was taken for cell morphology observation;LDH release in culture medium was measured using an LDH diagnostic kit;MTT analysis was used to assess the Glu-induced cytotoxicity.RESULTS:1)There were no differences in cellular morphology and cell growth characteristics between AQP4^+/+and AQP4^-/-astrocytes;2) Sustained exposure to Glu for 24 h caused damage to co-cultures of two genotypes and even worse lesion occurred in AQP4^-/-system;3)Less excitotoxicity was detected in primary cultured AQP4^-/-astrocytes, indicating severer loss of neurons in AQP4^-/-co-culture.CONCLUSION:Deficiency of AQP4 aggravates Glu-induced excitotoxicity in astrocyte-neuron co-cultures while decreases astrocytic susceptivity to Glu,suggesting that AQP4 is involved in excitotoxic damage to neurons and the functional impairment of astrocytes may be the key point. 2.Mechanisms involved in the increased excitotoxicity by AQP4 deficiencyAIM:To investigate the mechanisms of aggravated excitotoxicity by AQP4 knockout and to elucidate the regulative action of AQP4 on the astrocytic functions.METHODS:Primary astrocyte cultures were prepared from wildtype and homozygous mutant mice.RT-PCR and Western blotting were taken for glutamate transporters(GluTs)expression; [³H]-D,L-glutamate uptake analysis was performed for the assessment of GluTs function.RESULTS:1)Lack of AQP4 down-regulated astrocytic expression of glutamate transporter 1(GLT-1)but not of glutamate / aspartate transporter(GLAST);2)A lower uptake capability in AQP4^-/-astrocytes was detected.CONCLUSION:Deficiency of AQP4 down-regulates GLT-1 expression and Glu uptake in astrocytes,indicating AQP4 is actually involved in the modulation of astrocytes function and the mechanisms underlying the increased excitotoxicity may be the inhibition of Glu uptake.In summary,the present work provides direct evidences for the first time that AQP4 plays an important role in the process of IR and the underlying mechanisms predominantly lie in the modulation of GluTs function.The results obtained here substantially improve our understanding of AQP4-glutamanergic biology and intensively suggest:1) AQP4 is tightly involved in the pathology of stroke and plays an important role in the regulation of astroglial function;2)The therapeutic strategy targeted to astrocytic modulation with AQP4 may offer a new perspective for the development of new options for AIS treatment.