Role of Neutrophils in Exacerbation of Brain Injury After Focal Cerebral Ischemia in Hyperlipidemic Mice.
OBJECTIVE
Inflammation-related comorbidities contribute to stroke-induced immune responses and brain damage. We previously showed that hyperlipidemia exacerbates ischemic brain injury, which is associated with elevated peripheral and cerebral granulocyte numbers. Herein, we evaluate the contribution of neutrophils to the exacerbation of ischemic brain injury.


METHODS
Wild-type mice fed with a normal chow and ApoE knockout mice fed with a high cholesterol diet were exposed to middle cerebral artery occlusion. CXCR2 was blocked using the selective antagonist SB225002 (2 mg/kg) or neutralizing CXCR2 antiserum. Neutrophils were depleted using an anti-Ly6G antibody. At 72 hours post ischemia, immunohistochemistry, flow cytometry, and real-time polymerase chain reaction were performed to determine cerebral tissue injury and immunologic changes in the blood, bone marrow, and brain. Functional outcome was assessed by accelerated rota rod and tight rope tests at 4, 7, and 14 days post ischemia.


RESULTS
CXCR2 antagonization reduced neurological deficits and infarct volumes that were exacerbated in hyperlipidemic ApoE-/- mice. This effect was mimicked by neutrophil depletion. Cerebral neutrophil infiltration and peripheral neutrophilia, which were increased on ischemia in hyperlipidemia, were attenuated by CXCR2 antagonization. This downscaling of neutrophil responses was associated with increased neutrophil apoptosis and reduced levels of CXCR2, inducible nitric oxide synthase, and NADPH oxidase 2 expression on bone marrow neutrophils.


CONCLUSIONS
Our data demonstrate a role of neutrophils in the exacerbation of ischemic brain injury induced by hyperlipidemia. Accordingly, CXCR2 blockade, which prevents neutrophil recruitment into the brain, might be an effective option for stroke treatment in patients with hyperlipidemia.
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.). josephine.herz@uk-essen.de thorsten.doeppner@uk-essen.de.
Josephine
J
Herz
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.).
Pascal
P
Sabellek
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.).
Thomas E
TE
Lane
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.).
Matthias
M
Gunzer
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.).
Dirk M
DM
Hermann
From the Department of Neurology (J.H., P.S., D.M.H., T.R.D.), Department of Pediatrics I (J.H.), and Institute of Experimental Immunology and Imaging, University Duisburg-Essen (M.G.), University Hospital Essen, Essen, Germany; and Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City (T.E.L.).
Thorsten R
TR
Doeppner
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D002545:Brain Ischemia / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002791:Cholesterol, Dietary; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006949:Hyperlipidemias / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry; D020244:Infarction, Middle Cerebral Artery / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D007249:Inflammation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000276:immunology*; D010671:Phenylurea Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D023063:Receptors, Interleukin-8B / Q000037:antagonists & inhibitors / Q000276:immunology*
D001057:Apolipoproteins E; D002791:Cholesterol, Dietary; D008562:Membrane Glycoproteins; D010671:Phenylurea Compounds; D012333:RNA, Messenger; D023063:Receptors, Interleukin-8B; C112019:SB 225002; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases
CXCR2; brain injury; hyperlipidemia; inflammation; neutrophils; stroke