Portal de Periódicos da CAPES

Novel Docosanoids Inhibit Brain Ischemia-Reperfusion-mediated Leukocyte Infiltration and Pro-inflammatory Gene Expression

2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês

10.1074/jbc.m305841200

1083-351X

Victor L. Marcheselli, Song Hong, Walter J. Lukiw, Xiao Hua Tian, Karsten Gronert, Alberto E. Musto, Mattie Hardy, Juan Gimenez, Nan Chiang, Charles N. Serhan, Nicolás G. Bazán,

Cholesterol and Lipid Metabolism

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFκB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-β-induced NFκB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion. Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFκB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-β-induced NFκB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion. Brain ischemia-reperfusion triggers lipid peroxidation that participates in neural injury (1Beal M.F. Curr. Opin. Neurol. 1996; 6: 661-666Crossref Scopus (384) Google Scholar, 2Bazan N.G. Allan G. Ginsberg M.D. Bogousslavsky J. Cerebrovascular Disease: Pathophysiology, Diagnosis, and Management. Blackwell Science, Inc., Malden, MA1998: 532-555Google Scholar). Docosahexaenoic acid (DHA) 1The abbreviations used are: DHA, docosahexaenoic acid; HDHA, hydroperoxy-DHA; AP1, activator protein 1; HIF-1α, hypoxia-inducible factor 1-α; HN, human neural progenitor cells; MCA-O, middle cerebral artery occlusion; PMN, polymorphonuclear; IL, interleukin; STAT, signal transducers and activators of transcription; TTC, 2,3,5,-triphenyltetrazolium chloride; PBS, phosphate-buffered saline; LC, liquid chromatography; MS, mass spectrometry; PDA, photodiode array; ESI, electrospray ionization; SIM, selected ion monitoring.1The abbreviations used are: DHA, docosahexaenoic acid; HDHA, hydroperoxy-DHA; AP1, activator protein 1; HIF-1α, hypoxia-inducible factor 1-α; HN, human neural progenitor cells; MCA-O, middle cerebral artery occlusion; PMN, polymorphonuclear; IL, interleukin; STAT, signal transducers and activators of transcription; TTC, 2,3,5,-triphenyltetrazolium chloride; PBS, phosphate-buffered saline; LC, liquid chromatography; MS, mass spectrometry; PDA, photodiode array; ESI, electrospray ionization; SIM, selected ion monitoring. (22:6n-3) esterified in membrane phospholipids is released in brain ischemia (3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar, 4Yoshida S. Harik S. Busto R. Santiso M. Martinez E. Ginsberg M.D. J. Neurochem. 1984; 42: 711-717Crossref PubMed Scopus (36) Google Scholar) and is thought to yield lipid peroxides (5Roberts II, L.J. Montine T.J. Markesbery W.R. Tappert A.R. Hardy P. Chemtob S. Dettbarn W.D. Morrow J.D. J. Biol. Chem. 1998; 273: 13605-13612Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Leukocyte infiltration and pro-inflammatory gene expression are mediators of ischemic stroke damage (6Rothwell N.J. Luheshi G.N. Trends Neurosci. 2000; 23: 618-625Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 7Matsuo Y. Onodera H. Shiga Y. Nakamura M. Ninomiya M. Kihara T. Kogure K. Stroke. 1994; 25: 1469-1475Crossref PubMed Scopus (432) Google Scholar, 8Royo N.C. Wahl F. Stutzmann J.-M. Neuroreport. 1999; 10: 1363-1367Crossref PubMed Scopus (50) Google Scholar); however, there are no known messengers that down-regulate these events. The biosynthesis of oxygenated arachidonic acid messengers (9Gaudet R.J. Levine L. Stroke. 1980; 11: 648-652Crossref PubMed Scopus (47) Google Scholar, 10Moskovitz M.A. Kiwak K.J. Hekimian K. Levine L. Science. 1984; 224: 886-899Crossref PubMed Scopus (281) Google Scholar) triggered by cerebral ischemia-reperfusion is preceded by an early and rapid phospholipase A2 activation reflected in free arachidonic and docosahexaenoic acid accumulation (3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar, 4Yoshida S. Harik S. Busto R. Santiso M. Martinez E. Ginsberg M.D. J. Neurochem. 1984; 42: 711-717Crossref PubMed Scopus (36) Google Scholar, 11Aveldano M.I. Bazan N.G. Brain Res. 1975; 100: 99-110Crossref PubMed Scopus (66) Google Scholar). These fatty acids are released from membrane phospholipids where they are esterified (3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar). Both fatty acids are derived from dietary essential fatty acids; however, only DHA is concentrated in the central nervous system (12Bazan N.G. Wurtman R.J. Wurtman J.J. Nutrition and the Brain. Vol. 8. Raven Press, Ltd., New, York1990: 1-24Google Scholar). It is clear that synaptic membrane and retinal photoreceptor biogenesis is dependent on liver processing of the dietary DHA or of its precursor, linolenic acid, followed by blood lipoprotein transport (13Scott B.L. Bazan N.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2903-2907Crossref PubMed Scopus (379) Google Scholar). DHA is involved in memory formation (14Gamoh S. Hashimoto M. Sugioka K. Shahdat Hossain M. Hata N. Misawa Y. Masumura S. Neuroscience. 1999; 93: 237-241Crossref PubMed Scopus (255) Google Scholar), excitable membrane function (15McGahon B.M. Martin D.S. Horrobin D.F. Lynch M.A. Neuroscience. 1999; 94: 305-314Crossref PubMed Scopus (205) Google Scholar), photoreceptor cell biogenesis and function (16Gordon W.C. Bazan N.G. J. Neurosci. 1990; 10: 2190-2202Crossref PubMed Google Scholar), and neuronal signaling (17Mirnikjoo B. Brown S.E. Kim H.F. Marangell L.B. Sweatt J.D. Weeber E.J. J. Biol. Chem. 2001; 276: 10888-10896Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and has been implicated in neuroprotection (18Kim H.-Y. Akbar M. Lau A. Edsall L. J. Biol. Chem. 2000; 275: 35215-35223Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 19Lauritzen I. Blondeau N. Heurteaux C. Widmann C. Romey G. Lazdunski M. EMBO J. 2000; 19: 1784-1793Crossref PubMed Scopus (418) Google Scholar, 20Rodriguez de Turco E.B. Belayev L. Liu Y. Busto R. Parkins N. Bazan N.G. Ginsberg M.D. J. Neurochem. 2002; 83: 515-524Crossref PubMed Scopus (82) Google Scholar). Whether DHA itself or a DHA-derived messenger is involved in these events is not known. Moreover, to date, potent bioactive autacoids from DHA acting in nanomolar concentrations have not been identified in the central nervous system. Although certain docosanoids have been identified in the retina (21Bazan N.G. Birkle D.L. Reddy T.S. Biochem. Biophys. Res. Commun. 1984; 125: 741-747Crossref PubMed Scopus (95) Google Scholar) and have been proposed to be neuroprotective (12Bazan N.G. Wurtman R.J. Wurtman J.J. Nutrition and the Brain. Vol. 8. Raven Press, Ltd., New, York1990: 1-24Google Scholar), their physiologic properties have not been explored.To test the ability of the mouse brain to synthesize bioactive docosanoids, we used tandem LC-PDA-ESI-MS-MS-based lipidomic analysis in combination with ischemia-reperfusion. The rationale for use of this design was based upon the fact that brain ischemia releases unesterified DHA (3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar, 4Yoshida S. Harik S. Busto R. Santiso M. Martinez E. Ginsberg M.D. J. Neurochem. 1984; 42: 711-717Crossref PubMed Scopus (36) Google Scholar, 11Aveldano M.I. Bazan N.G. Brain Res. 1975; 100: 99-110Crossref PubMed Scopus (66) Google Scholar) and that during early stages of ischemia-reperfusion, endogenous signals of repair/neuroprotection may be generated.EXPERIMENTAL PROCEDURESReagents—Human recombinant IL-1β (14019) was from Sigma, and 10,17-diHDHA was prepared as described previously (22Hong S. Gronert K. Devchand P.R. Moussignac R.L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 23Serhan C.N. Clish C.B. Brannon J. Colgan S. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (930) Google Scholar). Normal human neural (HN) progenitor cells (CC-2599), neural progenitor maintenance medium, human epidermal and human fibroblast growth factors, gentamicin/amphotericin B (G/A1000), and neural survival factor-1 were obtained from Clonetics (Walkersville, MD). AP1, HIF-1α, NFκBp50/p65, and STAT-1α gel-shift consensus and mutant oligonucleotides were synthesized at the Louisiana State University Health Sciences Center core facility or were purchased from Promega Life Science (Madison, WI).Middle Cerebral Artery Occlusion (MCA-O) and Reperfusion—Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center, New Orleans, and followed the National Institutes of Health guidelines for experimental animal use. Mice (20–25 g body weight) were induced with 2% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. Anesthesia was maintained with 1% isoflurane. Temperature was maintained at 36.5–37.5 °C (Harvard homeothermic blanket). P-10 polyethylene catheters were placed in the femoral artery and vein, and blood pressure was monitored. Arterial blood was analyzed for PO2, PCO2, and pH after 1 h of ischemia and 30 min after the onset of reperfusion. The common carotid and external carotid arteries were temporarily ligated with a retracting suture, and the external carotid artery was dissected just proximal to its bifurcation. The occluding filament was introduced from the external carotid artery and advanced to the internal carotid artery. The arterial venous microclip was removed, and the filament was advanced so that the blunted tip lay in the anterior cerebral artery and the side of the filament occluded the origin of the MCA. The stump of the external carotid artery was ligated, and tension on the retracting suture to the common carotid artery was gently released, restoring blood flow to the carotid system. The animals were allowed to recover from anesthesia, and 15 min before the start of reperfusion, a neurologic performance test was conducted (24Bederson J.B. Pitts L.H. Tsuji M. Nishimura M.C. Davis R.L. Bartkowski H. Stroke. 1986; 17: 472-476Crossref PubMed Scopus (2400) Google Scholar). This neurologic assessment allowed definition of the degree of damage. A rating of Class III or Class IV indicated very severe damage; Class I indicated very mild damage. Thus, to enhance consistency in the group of selected animals, only mice rated as having Class II neurologic performance were included in the study. The variables minimized by this screening are anatomic variants of brain arteries and surgical procedure. About 85% of the mice had Class II neurologic performance. Occlusion of the middle cerebral artery was maintained for 1 h; reperfusion was then established by delicate retrieval of the occluding suture from the arterial lumen, restoring blood flow to the region of the MCA. The animals were killed by decapitation, and the hippocampi and brain cortices were rapidly dissected and frozen. Samples were kept at -80 °C until analysis. In some experiments, aspirin (7.5 mg/kg) was administered by gavage 15 min before MCA-O.Assessment of Stroke Volume—Forty-eight hours after MCA-O, the mice were killed, and their brains were dissected and immersed in ice-cold saline. The brains were embedded in agar blocks and sectioned into coronal slices 1 mm thick by a vibratome (Vibratome Co., St. Louis, MO). The sections were incubated at room temperature in a 3% buffered solution of 2,3,5,-triphenyltetrazolium chloride (TTC). Once the color had developed (10–15 min), sections were fixed in 10% buffered formalin and kept at 4 °C until images were recorded by a camera (Cool-snap, Nikon) mounted to a dissecting microscope. Digital images were analyzed, and total and stroke areas were calculated and analyzed by Adobe Photoshop software. Serial sections were made for all animals.Human Neural (HN) Progenitor Cells in Primary Culture—HN cells were grown to ∼70% confluence (∼50,000 cells per 3.5-cm diameter well) in 6-well culture plates (Costar) at 37 °C, 5% CO2, 20% O2, 75% N2 in humidified air at 1 atmosphere (normoxic conditions) in neural progenitor maintenance medium (Clonetics CC-4241) supplemented with human fibroblast growth factor, neural survival factor-1, human epidermal growth factor, and GA-1000 as described by the manufacturer (Clonetics, Walkersville, MD). HN cells tested negative for HIV-1, hepatitis B and C, mycoplasma, bacteria, yeast, and fungi and positive for the glial and neuronal markers glial fibrillary acidic protein, mitogen-activated protein 2, and β-tubulin III (Clonetics). After 2 weeks of development, HN cells were exposed to human recombinant IL-1β (10 ng/ml) for 3 h in the presence or absence of 0.03, 0.3, 3.0, 30, and 300 nm 10,17S-docosatriene, DHA, or PBS, pH 7.4 (control). RNA and protein were rapidly isolated using Trizol Reagent (Invitrogen, Carlsbad, CA) and stored at -81 °C within minutes of isolation.COX-2 RNA Abundance in Hippocampus and HN Cells—Abundance of human-specific COX-1 and COX-2 RNA message was assayed using reverse transcriptase PCR (25Bazan N.G. Lukiw W.J. J. Biol. Chem. 2002; 277: 30359-30367Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).Electrophoretic Mobility Shift Assay of Human AP1, HIF-1α, NFκBp50/p65, and STAT-1α—Nuclear protein extracts were prepared from one to three 3.5-cm diameter wells of HN cells and quantitated. Nuclear protein extracts (5 μg) derived from HN cells were incubated with [γ-32P]ATP (∼3000 Ci/mmol)-end-labeled AP1, HIF-1α, NFκBp50/p65, or STAT-1α consensus and mutant oligonucleotides in 5-μl volumes, reacted for 30 min on ice, analyzed on 5 or 10% acrylamide, 90 mm Tris borate, pH 8.4, 1 mm EDTA gels, dried onto 2-mm Whatman filter paper at 80 °C for 2 h, and phosphorimaged using a Typhoon Variable Mode Imager (Amersham Biosciences).Polymorphonuclear (PMN) Leukocyte Infiltration Measurement by the Myeloperoxidase Assay—Inhibition of leukocyte infiltration by 10,17S-docosatriene or by DHA was measured in mouse hippocampi and neocortices after 1 h of MCA-O and 48 h of reperfusion. Lipids were delivered in the indicated quantities for each experiment by perfusion (250 nl/h) through Alzet mini-pumps implanted into the third ventricle. Hippocampi and neocortices from the ipsilateral (MCA-O-reperfusion) and contralateral sides were rapidly dissected. The brain samples were assayed for myeloperoxidase activity (26Huang J. Choudhri T.F. Winfree C.J. McTaggart R.A. Kiss S. Mocco J. Kim L.J. Protopsaltis T.S. Zhang Y. Pinsky D.J. Connolly Jr., E.S. Stroke. 2000; 31: 3047-3053Crossref PubMed Scopus (128) Google Scholar). Briefly, the brain tissues were homogenized in 10 mm phosphate buffer (pH 7.4) and then frozen and thawed with liquid N2 followed by sonication. Samples were precipitated at 10,000 × g for 10 min, and then aliquots of supernatants were added to a 10-mm phosphate buffer (pH 6.0) and a substrate solution containing O-dianisidine (Sigma) and 0.025% hydrogen peroxide and finally incubated at 37 °C for 45 min. Spectrophotometric detection was obtained at 460 nm.Immunohistochemistry of Myeloperoxidase to Assess Leukocyte Infiltration—After MCA-O, mice were infused by Alzet mini-pumps with vehicle or 10,17S-docosatriene. After 48 h, the mice were anesthetized and killed by intracardial perfusion of ice-cold saline followed by 10% neutral buffered formalin. The brain tissues were allowed to equilibrate overnight in 4% buffered formalin followed by 30% sucrose in 0.1 m PBS. Frozen sections were made at a thickness of 10 μm and mounted on glass slides. The sections were permeabilized with 0.6% Triton X-100 for 10 min, washed in PBS, and blocked in 2% goat serum in PBS for 30 min. Incubation with myeloperoxidase/fluorescein isothiocyanate-conjugated antibody (Dako A/S) was performed at 1:200 dilution for 2 h. The sections were washed in Tween 20 in PBS and then mounted in Vectashield (Vector, CA). Images were recorded by deconvolution microscope (Intelligent Imaging Innovations, Denver, CO).LC-MS-MS Analysis of Docosanoids—Quantitative analysis of docosanoids by LC-MS-MS was performed in hippocampi from mice (C57/BL-6, 20–25 g body weight) killed by head-focused microwave radiation at different time points after the onset of reperfusion. The hippocampi were rapidly dissected (20–70 mg wet tissue weight), homogenized in cold methanol, and kept under nitrogen at -80 °C until purification. Purification was performed by solid-phase extraction technique (27Gronert K. Clish C.B. Romano M. Serhan C.N. Methods Mol. Biol. 1999; 120: 119-144PubMed Google Scholar). In short, samples pre-equilibrated at pH 3.0 were loaded onto C18 columns (Varian) and eluted with 10 ml of 1% methanol in ethyl acetate (EM Science). Samples were concentrated by a nitrogen stream evaporator before LC-MS analysis. Samples were loaded into a Surveyor MS pump (Thermo-Finnegan) equipped with a C18 discovery column (Supelco), 10 cm × 2.1 mm inner diameter, 5 μm internal phase. Samples were eluted in a linear gradient (100% solution A (60:40:0.01 methanol/water/acetic acid) to 100% solution B (99.99:0.01 methanol/acetic acid)) and run at a flow rate of 300 μl/min for 45 min. LC effluents were diverted to an electrospray ionization probe (ESI) on a TSQ Quantum (Thermo-Finnegan) triple quadrupole mass spectrometer running on negative ion detection mode. Docosanoid standards were used for calibration and optimization. The instrument was run on full-scan mode to detect parent ions and selected reaction monitoring for quantitative analysis to detect daughter ions simultaneously. The selected parent ions were 327 for DHA and 359 for 10,17S-docosatriene. Moreover, daughter ions were 325.1 and 297, respectively.RESULTSBrain Ischemia-Reperfusion Triggers the Synthesis of Docosahexaenoic Acid-Oxygenation Pathways—We used 1 h of right middle cerebral artery occlusion in mice followed by reperfusion to assess the formation of docosahexaenoic acid-oxygenation derivatives. Under these conditions there is active release of free docosahexaenoic acid from brain membrane phospholipids (2Bazan N.G. Allan G. Ginsberg M.D. Bogousslavsky J. Cerebrovascular Disease: Pathophysiology, Diagnosis, and Management. Blackwell Science, Inc., Malden, MA1998: 532-555Google Scholar, 3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar, 4Yoshida S. Harik S. Busto R. Santiso M. Martinez E. Ginsberg M.D. J. Neurochem. 1984; 42: 711-717Crossref PubMed Scopus (36) Google Scholar). This model of transient focal ischemia greatly affects the hippocampus, a brain region rich in neurons vulnerable in ischemic stroke and in other neurologic diseases (1Beal M.F. Curr. Opin. Neurol. 1996; 6: 661-666Crossref Scopus (384) Google Scholar, 2Bazan N.G. Allan G. Ginsberg M.D. Bogousslavsky J. Cerebrovascular Disease: Pathophysiology, Diagnosis, and Management. Blackwell Science, Inc., Malden, MA1998: 532-555Google Scholar).Fig. 1A shows the time course of formation of novel docosanoids in the ipsilateral hippocampus during ischemia-reperfusion. There was generation of 17-hydroperoxy-DHA, the 15-lipoxygenase-like action on DHA (22Hong S. Gronert K. Devchand P.R. Moussignac R.L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar), and a novel 10,17S-docosatriene accumulated up to 8 h of reperfusion. The MS-MS spectrum of 10,17S-docosatriene (Fig. 1, B and C) corresponded to a dihydroxy-containing DHA with prominent fragment ions at m/z 359 (Fig. 1C, [M-H]). There were also fragment ions at m/z 323 ([M-H]-2H2O), 315 ([M-H]-CO2), 297 ([M-H]-H2O-CO2), and 277 ([M-H]-2H2O-CO2-2H). Other diagnostic ions were essentially identical to those recently documented (22Hong S. Gronert K. Devchand P.R. Moussignac R.L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar) in murine brain, human blood, and glial cells. In addition, there was a time-dependent formation of the carbon 22-ω hydroxylation product, 4,17di-HDHA (Fig. 1A) as determined by analysis (at m/z 375) of the MS-MS spectrum (Fig. 1D).Because aspirin is often used prophylactically as well as therapeutically to manage cerebrovascular diseases, we next asked whether brain biosynthesis of DHA messengers is modified in the presence of aspirin in vivo. Moreover, in non-neural tissues, aspirin triggers the biosynthesis of anti-inflammatory lipid mediators (23Serhan C.N. Clish C.B. Brannon J. Colgan S. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (930) Google Scholar, 28Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R.L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1316) Google Scholar). We examined the formation of DHA-derived docosanoids in the presence of aspirin during reperfusion after an ischemic stroke (Fig. 1). Fig. 1E shows the time course of docosanoid formation with aspirin treatment. There was a shift away from the products generated from endogenous sources of DHA in the absence of aspirin (Fig. 1A) toward products that include the novel 17R-series resolvins, in particular 7,17R-diHDHA and 7,8,17R-triHDHA, which were formed and were present at the earliest time intervals. There was a marked accumulation in 17R-HDHA, shown recently to be a product of aspirin-acetylated cyclooxygenase-2 (28Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R.L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1316) Google Scholar).Fig. 1, F and G, shows a representative LC-MS-MS of the ipsilateral hippocampus following treatment with aspirin and depicts the chromatographic profile of products identified via MS-MS and lipidomic analyses. Here we also confirmed the release of DHA under these conditions (3Bazan N.G. Biochim. Biophys. Acta. 1970; 218: 1-10Crossref PubMed Scopus (682) Google Scholar, 4Yoshida S. Harik S. Busto R. Santiso M. Martinez E. Ginsberg M.D. J. Neurochem. 1984; 42: 711-717Crossref PubMed Scopus (36) Google Scholar, 11Aveldano M.I. Bazan N.G. Brain Res. 1975; 100: 99-110Crossref PubMed Scopus (66) Google Scholar) (Fig. 1H). To assess that the aspirin dose administered by gavage did reach the brain, the time courses for prostaglandin E2, leukotriene B4, and lipoxin A4 production were determined in the hippocampi in parallel assays (Fig. 2, PGE 2, LTB 4, and LXA 4). The rapid accumulation of prostaglandin E2 within 8 h of reperfusion was inhibited by aspirin treatment. Maximal leukotriene B4 was generated in aspirin-treated hippocampi by 8 h, as was the case with lipoxin A4, which also peaked within 8 h. Together these results clearly indicate that the dose of aspirin used in vivo did access the central nervous system.Fig. 2Time course of prostaglandin E2, leukotriene B4, and lipoxin A4 formation in mouse hippocampus after 1 h of MCA-O followed by reperfusion with or without aspirin treatment. Detection was performed by sensitive and specific enzyme-linked immunosorbent assay, in tandem, for prostaglandin E2 (PGE 2), leukotriene B4 (LTB 4), and lipoxins (LXA 4) (Neogen, Lexington, KY). This study showed inhibitory changes in the eicosanoids when animals were pretreated with aspirin (15 min) before MCA-O. C57BL/6 mice were treated by gavages with vehicle (sterile saline) or aspirin (7.52 mg/kg).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3 depicts the proposed pathway to generate the novel 10,17S-diHDHA ω-oxidation product measured in the absence of aspirin. In our experiments, 4,17S-diHDHA did show bioactivity in PMN infiltration, but these were less than those evoked by equimolar concentrations of 10,17S-docosatriene in a non-neural experimental model (Fig. 4). Fig. 4, therefore, confirms that 4,17S-diHDHA inhibits PMN leukocyte exudate formation in murine peritonitis models (28Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R.L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1316) Google Scholar). This ω-oxidation product is the likely inactivation product for 10,17S-diHDHA. Fig. 3 also depicts the resolvin or 17R series pathways. These are the products formed in the presence of aspirin.Fig. 3Proposed biosynthetic pathways for 10,17S-docosatriene and the aspirin-triggered 17R-series resolvins. The stereochemistry for compounds in both pathways is based on the biogenic total synthesis, lipidomic analyses, and alcohol-trapping profiles (22Hong S. Gronert K. Devchand P.R. Moussignac R.L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 23Serhan C.N. Clish C.B. Brannon J. Colgan S. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (930) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Inhibition of leukocyte infiltration in zymosan-induced peritonitis by 4,17S-diHDHA and 10,17S-docosatriene. The 4,17S-diHDHA caused dose-dependent inhibition of polymorphonuclear leukocyte infiltration. Here 100-ng 10,17S-docosatriene caused potent inhibition. Peritonitis was induced in 6–8-week-old male FVB mice (Charles River Laboratories) by peritoneal injection of 1 mg of zymosan A. Compounds 4,17S- and 10,17S-diHDHA were injected by intravenous bolus injection, 1.5 min before zymosan A treatment. Two h after induction of peritonitis, rapid peritoneal lavages were collected, and cell type enumeration was performed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Polymorphonuclear Leukocyte Infiltration Mediated by Focal Ischemic Stroke in Mice Is Inhibited by 10,17S-Docosatriene—We monitored PMN infiltration, a major factor in mediating brain ischemia-reperfusion damage (7Matsuo Y. Onodera H. Shiga Y. Nakamura M. Ninomiya M. Kihara T. Kogure K. Stroke. 1994; 25: 1469-1475Crossref PubMed Scopus (432) Google Scholar, 8Royo N.C. Wahl F. Stutzmann J.-M. Neuroreport. 1999; 10: 1363-1367Crossref PubMed Scopus (50) Google Scholar, 29Chopp M. Li Y. Zhang R.L. Prostak J. J. Cereb. Blood Flow Metab. 1996; 16: 578-584Crossref PubMed Scopus (174) Google Scholar, 30Chatzipanteli K. Alonso O.F. Kraydieh S. Dietrich W.D. J. Cereb. Blood Flow Metab. 2000; 20: 531-542Crossref PubMed Scopus (148) Google Scholar). PMN infiltration is a complex multistep process that is modulated by the coordinated expression of adhesion and signaling molecules (26Huang J. Choudhri T.F. Winfree C.J. McTaggart R.A. Kiss S. Mocco J. Kim L.J. Protopsaltis T.S. Zhang Y. Pinsky D.J. Connolly Jr., E.S. Stroke. 2000; 31: 3047-3053Crossref PubMed Scopus (128) Google Scholar). DHA-derived messengers were very recently reported to inhibit PMN invasion outside of the central nervous system in the air-pouch model (23Serhan C.N. Clish C.B. Brannon J. Colgan S. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (930) Google Scholar, 26Huang J. Choudhri T.F. Winfree C.J. McTaggart R.A. Kiss S. Mocco J. Kim L.J. Protopsaltis T.S. Zhang Y. Pinsky D.J. Connolly Jr., E.S. Stroke. 2000; 31: 3047-3053Crossref PubMed Scopus (128) Google Scholar, 28Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R.L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1316) Google Scholar).To determine whether 10,17S-docosatriene displays counter-active bioactivity during brain ischemia-reperfusion-induced PMN infiltration, this docosanoid was constantly infused into the third ventricle during 48 h of superfusion in the amounts shown in Fig. 5. By immunostaining of myeloperoxidase as a marker o