ABSTRACT
Stimulation of Na+ /H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/- ;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/- ;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.
Subject(s)
Astrocytes/metabolism , Blood-Brain Barrier/pathology , Gliosis/pathology , Infarction, Middle Cerebral Artery/complications , Sodium-Hydrogen Exchanger 1/deficiency , Animals , Astrocytes/ultrastructure , Blood-Brain Barrier/ultrastructure , Brain Infarction/diagnosis , Brain Infarction/etiology , Brain Infarction/genetics , Cerebrovascular Circulation/genetics , Cerebrovascular Circulation/physiology , Disease Models, Animal , Gene Expression Regulation/genetics , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Gliosis/etiology , Gliosis/genetics , Gliosis/metabolism , Infarction, Middle Cerebral Artery/pathology , Male , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Transmission , Motor Activity/genetics , Neurologic Examination , Reperfusion , Sodium-Hydrogen Exchanger 1/geneticsABSTRACT
The WNK-SPAK kinase signaling pathway controls renal NaCl reabsorption and systemic blood pressure by regulating ion transporters and channels. A WNK3-SPAK complex is highly expressed in brain, but its function in this organ remains unclear. Here, we investigated the role of this kinase complex in brain edema and white matter injury after ischemic stroke. Wild-type, WNK3 knockout, and SPAK heterozygous or knockout mice underwent transient middle cerebral artery occlusion. One cohort of mice underwent magnetic resonance imaging. Ex-vivo brains three days post-ischemia were imaged by slice-selective spin-echo diffusion tensor imaging magnetic resonance imaging, after which the same brain tissues were subjected to immunofluorescence staining. A second cohort of mice underwent neurological deficit analysis up to 14 days post-transient middle cerebral artery occlusion. Relative to wild-type mice, WNK3 knockout, SPAK heterozygous, and SPAK knockout mice each exhibited a >50% reduction in infarct size and associated cerebral edema, significantly less demyelination, and improved neurological outcomes. We conclude that WNK3-SPAK signaling regulates brain swelling, gray matter injury, and demyelination after ischemic stroke, and that WNK3-SPAK inhibition has therapeutic potential for treating malignant cerebral edema in the setting of middle cerebral artery stroke.
Subject(s)
Brain Edema/genetics , Brain/pathology , Gene Deletion , Infarction, Middle Cerebral Artery/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Brain/metabolism , Brain Edema/etiology , Brain Edema/pathology , Female , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/genetics , Myelin Sheath/metabolism , Myelin Sheath/pathology , White Matter/metabolism , White Matter/pathologyABSTRACT
Platelets are anucleated cell fragments derived from mature megakaryocytes and function in hemostasis when the endothelium is injured. Hemostasis involving platelets can be divided into four phases: adhesion, activation, secretion, and aggregation. Platelet activation requires a rise in intracellular Ca(2+) concentrations and results in both a morphological change and the secretion of platelet granule contents. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates the intracellular pH (pHi) and the volume of platelets. In addition, NHE1 plays a large role in platelet activation. Thrombus generation involves NHE1 activation and an increase in [Ca(2+)]i, which results from NHE1-mediated Na(+) overload and the reversal of the Na(+)/Ca(2+) exchanger. Cariporide (HOE-642), a potent NHE1 inhibitor, has inhibitory effects on the degranulation of human platelets, the formation of platelet-leukocyte-aggregates, and the activation of the GPIIb/IIIa receptor (PAC-1). However, despite the demonstrated protection against myocardial infarction as mediated by cariporide in patients undergoing coronary artery bypass graft surgery, the EXPEDITION clinical trial revealed that cariporide treatment increased mortality due to thromboembolic stroke. These findings suggest that a better understanding of NHE1 and its effect on platelet function and procoagulant factor regulation is warranted in order to develop therapies using NHE inhibitors.
