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Brain 2-Arachidonoylglycerol Levels Are Dramatically and Rapidly Increased Under Acute Ischemia-Injury Which Is Prevented by Microwave Irradiation

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Lipids

Abstract

The involvement of brain 2-arachidonoylglycerol (2-AG) in a number of critical physiological and pathophysiological regulatory mechanisms highlights the importance for an accurate brain 2-AG determination. In the present study, we validated head-focused microwave irradiation (MW) as a method to prevent postmortem brain 2-AG alterations before analysis. We compared MW to freezing to prevent 2-AG induction and estimated exogenous and endogenous 2-AG stability upon exposure to MW. Using MW, we measured, for the first time, true 2-AG brain levels under basal conditions, 30 s after brain removal from the cranium, and upon exposure to 5 min of brain global ischemia. Our data indicate that brain 2-AG levels are instantaneously and dramatically increased approximately 60-fold upon brain removal from the cranium. With 5 min of brain global ischemia 2-AG levels are also, but less dramatically, increased 3.5-fold. Our data indicate that brain tissue fixation with MW is a required technique to measure both true basal 2-AG levels and 2-AG alterations under different experimental conditions including global ischemia, and 2-AG is stable upon exposure to MW.

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Abbreviations

2-AG:

2-Arachidonoylglycerol

2-AGd8 :

2-Arachidonoylglycerol containing deuterium atoms at the 5, 6, 8, 9, 11, 12, 14, and 15 positions of the arachidonic acid moiety

AEA:

N-Arachidonoylethanolamine

aCSF:

Artificial cerebral-spinal fluid

DAG:

Diacylglycerols

Lyso-PLC:

Lysophosphatidylinositol-selective PLC

MAGL:

Mono-acylglycerol lipase

MW:

Microwave irradiation

non-MW:

Non-fixed tissue

PLC:

Phospholipase C

PtdIns:

Phosphatidylinositol

Lyco-PLC:

Lysophosphatidylinositol-selective PLC

References

  1. Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R, Krey JF, Walker JM, Holmes PV, Crystal JD, Duranti A, Tontini A, Mor M, Tarzia G, Piomelli D (2005) An endocannabinoid mechanism for stress-induced analgesia. Nature 435:1108–1112

    Article  CAS  PubMed  Google Scholar 

  2. Nyilas R, Gregg LC, MacKie K, Watanabe M, Zimmer A, Hohmann AG, Katona I (2009) Molecular architecture of endocannabinoid signaling at nociceptive synapses mediating analgesia. Eur J Neurosci 29:1964–1978

    Article  PubMed  PubMed Central  Google Scholar 

  3. Flores Á, Maldonado R, Berrendero F (2013) Cannabinoid-hypocretin cross-talk in the central nervous system: what we know so far. Front Neurosci. doi:10.3389/fnins.2013.00256

    PubMed  PubMed Central  Google Scholar 

  4. Kreitzer AC, Malenka RC (2007) Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson’s disease models. Nature 445:643–647

    Article  CAS  PubMed  Google Scholar 

  5. Ruehle S, Rey AA, Remmers F, Lutz B (2012) The endocannabinoid system in anxiety, fear memory and habituation. J Psychopharmacol (Oxf) 26:23–39

    Article  CAS  Google Scholar 

  6. Jenniches I, Ternes S, Albayram O, Otte DM, Bach K, Bindila L, Michel K, Lutz B, Bilkei-Gorzo A, Zimmer A (2015) Anxiety, Stress, and Fear Response in Mice with Reduced Endocannabinoid Levels. Biol Psychiatry. doi:10.1016/j.biopsych.2015.03.033

    Google Scholar 

  7. Melis M, Pillolla G, Bisogno T, Minassi A, Petrosino S, Perra S, Muntoni AL, Lutz B, Gessa GL, Marsicano G, Di Marzo V, Pistis M (2006) Protective activation of the endocannabinoid system during ischemia in dopamine neurons. Neurobiol Dis 24:15–27

    Article  CAS  PubMed  Google Scholar 

  8. Pellegrini-Giampietro DE, Mannaioni G, Bagetta G (2009) Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death. FEBS J 276:2–12

    Article  CAS  PubMed  Google Scholar 

  9. Amantea D, Spagnuolo P, Bari M, Fezza F, Mazzei C, Tassorelli C, Morrone LA, Corasaniti MT, Maccarrone M, Bagetta G (2007) Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17β-estradiol. FEBS J 274:4464–4775

    Article  CAS  PubMed  Google Scholar 

  10. Degn M, Lambertsen KL, Petersen G, Meldgaard M, Artmann A, Clausen BH, Hansen SH, Finsen B, Hansen HS, Lund TM (2007) Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice. J Neurochem 103:1907–1916

    Article  CAS  PubMed  Google Scholar 

  11. Panikashvili D, Simeonidou C, Ben-Shabat S, Hanus L, Breuer A, Mechoulam R, Shohami E (2001) An endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature 413:527–531

    Article  CAS  PubMed  Google Scholar 

  12. Cristino L, Busetto G, Imperatore R, Ferrandino I, Palomba L, Silvestri C, Petrosino S, Orlando P, Bentivoglio M, Mackie K, Di Marzo V (2013) Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons. Proc Natl Acad Sci USA 110:E2229–E2238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Di Marzo V, Hill MP, Bisogno T, Crossman AR, Brotchie JM (2000) Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson’s disease. FASEB J 14:1432–1438

    Article  PubMed  Google Scholar 

  14. Baker D, Pryce G, Croxford JL, Brown P, Pertwee RG, Makriyannis A, Khanolkar A, Layward L, Fezza F, Bisogno T, Di Marzo V (2000) Endocannabinoids control spasticity in a multiple sclerosis model. FASEB J 15(2):300–302

    PubMed  Google Scholar 

  15. Földy C, Neu A, Jones MV, Soltesz I (2006) Presynaptic, activity-dependent modulation of cannabinoid type 1 receptor-mediated inhibition of GABA Release. J Neurosci 26:1465–1469

    Article  PubMed  Google Scholar 

  16. Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M (2009) Endocannabinoid-mediated control of synaptic transmission. Physiol Rev 89:309–380

    Article  CAS  PubMed  Google Scholar 

  17. Melis M, Perra S, Muntoni AL, Pillolla G, Lutz B, Marsicano G, Di Marzo V, Gessa GL, Pistis M (2004) Prefrontal cortex stimulation induces 2-arachidonoyl-glycerol-mediated suppression of excitation in dopamine neurons. J Neurosci 24:10707–10715

    Article  CAS  PubMed  Google Scholar 

  18. Guindon J, Hohmann AG (2009) The endocannabinoid system and pain. CNS Neurol Disord Drug Targets 8:403–421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rea K, Roche M, Finn DP (2007) Supraspinal modulation of pain by cannabinoids: the role of GABA and glutamate. Br J Pharmacol 152:633–648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Baur R, Kielar M, Richter L, Ernst M, Ecker GF, Sigel E (2013) Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the β2 subunit of GABAA receptors. J Neurochem 126:29–36

    Article  CAS  PubMed  Google Scholar 

  21. Sigel E, Baur R, Rácz I, Marazzi J, Smart TG, Zimmer A, Gertsch J (2011) The major central endocannabinoid directly acts at GABA A receptors. Proc Natl Acad Sci USA 108:18150–18155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rouzer CA, Marnett LJ (2011) Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways. Chem Rev 111:5899–5921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nomura DK, Morrison BE, Blankman JL, Long JZ, Kinsey SG, Marcondes MCG, Ward AM, Hahn YK, Lichtman AH, Conti B, Cravatt BF (2011) Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science 334:809–813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Maccarrone M, Attinà M, Cartoni A, Bari M, Finazzi-Agrò A (2001) Gas chromatography–mass spectrometry analysis of endogenous cannabinoids in healthy and tumoral human brain and human cells in culture. J Neurochem 76:594–601

    Article  CAS  PubMed  Google Scholar 

  25. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K (1995) 2-Arachidonoylgylcerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 215:89–97

    Article  CAS  PubMed  Google Scholar 

  26. Sugiura T, Yoshinaga N, Kondo S, Waku K, Ishima Y (2000) Generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in picrotoxinin-administered rat brain. Biochem Biophys Res Commun 271:654–658

    Article  CAS  PubMed  Google Scholar 

  27. Sugiura T, Yoshinaga N, Waku K (2001) Rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain after decapitation. Neurosci Lett 297:175–178

    Article  CAS  PubMed  Google Scholar 

  28. Stella N, Schweitzer P, Plomelli D (1997) A second endogenous’ cannabinoid that modulates long-term potentiation. Nature 388:773–778

    Article  CAS  PubMed  Google Scholar 

  29. Bisogno T, Berrendero F, Ambrosino G, Cebeira M, Ramos JA, Fernandez-Ruiz JJ, Di Marzo V (1999) Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function. Biochem Biophys Res Commun 256:377–380

    Article  CAS  PubMed  Google Scholar 

  30. Kathuria S, Gaetani S, Fegley D, Valino F, Duranti A, Tontini A, Mor M, Tarzia G, Rana GL, Calignano A, Giustino A, Tattoli M, Palmery M, Cuomo V, Piomelli D (2003) Modulation of anxiety through blockade of anandamide hydrolysis. Nat Med 9:76–81

    Article  CAS  PubMed  Google Scholar 

  31. Ferrer B, Asbrock N, Kathuria S, Piomelli D, Giuffrida A (2003) Effects of levodopa on endocannabinoid levels in rat basal ganglia: implications for the treatment of levodopa-induced dyskinesias. Eur J Neurosci 18:1607–1614

    Article  PubMed  Google Scholar 

  32. Hansen HH, Schmid PC, Bittigau P, Lastres-Becker I, Berrendero F, Manzanares J, Ikonomidou C, Schmid HHO, Fernández-Ruiz JJ, Hansen HS (2001) Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration. J Neurochem 78:1415–1427

    Article  CAS  PubMed  Google Scholar 

  33. Kondo S, Kondo H, Nakane S, Kodaka T, Tokumura A, Waku K, Sugiura T (1998) 2-Arachidonoylglycerol, an endogenous cannabinoid receptor agonist: identification as one of the major species of monoacylglycerols in various rat tissues, and evidence for its generation through Ca2+ -dependent and -independent mechanisms. FEBS Lett 429:152–156

    Article  CAS  PubMed  Google Scholar 

  34. Chen Y, Nie H, Tian L, Tong L, Yang L, Lao N, Dong H, Sang H, Xiong L (2013) Nicotine-induced neuroprotection against ischemic injury involves activation of endocannabinoid system in rats. Neurochem Res 38:364–370

    Article  CAS  PubMed  Google Scholar 

  35. Kingsley PJ, Marnett LJ (2003) Analysis of endocannabinoids by Ag+ coordination tandem mass spectrometry. Anal Biochem 314:8–15

    Article  CAS  PubMed  Google Scholar 

  36. Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG, Hermann H, Tang J, Hofmann C, Zieglgansberger W, Di Marzo V, Lutz B (2002) The endogenous cannabinoid system controls extinction of aversive memories. Nature 418:530–534

    Article  CAS  PubMed  Google Scholar 

  37. Watanabe S, Doshi M, Hamazaki T (2003) n-3 Polyunsaturated fatty acid (PUFA) deficiency elevates and n-3 PUFA enrichment reduces brain 2-arachidonoylglycerol level in mice. Prostaglandins Leukot Essent Fatty Acids 69:51–59

    Article  CAS  PubMed  Google Scholar 

  38. Hanuš L, Avraham Y, Ben-Shushan D, Zolotarev O, Berry EM, Mechoulam R (2003) Short-term fasting and prolonged semistarvation have opposite effects on 2-AG levels in mouse brain. Brain Res 983:144–151

  39. Franklin A, Parmentier-Batteur S, Walter L, Greenberg DA, Stella N (2003) Palmitoylethanolamide increases after focal cerebral ischemia and potentiates microglial cell motility. J Neurosci 23:7767–7775

    CAS  PubMed  Google Scholar 

  40. Van Der Stelt M, Veldhuis WB, Van Haaften GW, Fezza F, Bisogno T, Bär PR, Veldink GA, Vliegenthart JFG, Di Marzo V, Nicolay K (2001) Exogenous anandamide protects rat brain against acute neuronal injury in vivo. J Neurosci 21:8765–8771

    PubMed  Google Scholar 

  41. Muthian S, Rademacher DJ, Roelke CT, Gross GJ, Hillard CJ (2004) Anandamide content is increased and CB1 cannabinoid receptor blockade is protective during transient, focal cerebral ischemia. Neuroscience 129:743–750

    Article  CAS  PubMed  Google Scholar 

  42. Ueda H, Kobayashi T, Kishimoto M, Tsutsumi T, Okuyama H (1993) A possible pathway of phosphoinositide metabolism through EDTA-insensitive phospholipase A1 followed by lysophosphoinositide-specific phospholipase C in rat brain. J Neurochem 61:1874–1881

    Article  CAS  PubMed  Google Scholar 

  43. Blankman JL, Simon GM, Cravatt BF (2007) A Comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol 14:1347–1356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vila A, Rosengarth A, Piomelli D, Cravatt B, Marnett LJ (2007) Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase. Biochemistry 46:9578–9585

    Article  CAS  PubMed  Google Scholar 

  45. Blankman JL, Cravatt BF (2013) Chemical probes of endocannabinoid metabolism. Pharmacol Rev 65:849–871

    Article  PubMed  PubMed Central  Google Scholar 

  46. Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL, Kathuria S, Piomelli D (2002) Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci USA 99:10819–10824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nithipatikom K, Endsley MP, Isbell MA, Wheelock CE, Hammock BD, Campbell WB (2005) A new class of inhibitors of 2-arachidonoylglycerol hydrolysis and invasion of prostate cancer cells. Biochem Biophys Res Commun 332:1028–1033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ikeda M, Yoshida S, Busto R, Santiso M, Ginsberg MD (1986) Polyphosphoinositides as a probable source of brain free fatty acids accumulated at the onset of ischemia. J Neurochem 47:123–132

    Article  CAS  PubMed  Google Scholar 

  49. Yoshida S, Ideda M, Busto R, Santiso M, Martinez E, Ginsberg MD (1986) Cerebral phosphoinositide, triacylglycerol, and energy metabolism in reversible ischemia: origin and fate of free fatty acids. J Neurochem 47:744–757

    Article  CAS  PubMed  Google Scholar 

  50. Lin TN, Liu TH, Xu J, Hsu CY, Sun GY (1991) Brain polyphosphoinositide metabolism during focal ischemia in rat cortex. Stroke 22:495–498

    Article  CAS  PubMed  Google Scholar 

  51. Murphy EJ, Haun SE, Rosenberger TA, Horrocks LA (1995) Altered lipid metabolism in the presence and absence of extracellular Ca 2+ during combined oxygen-glucose deprivation in primary astrocyte cell cultures. J Neurosci Res 42:109–116

    Article  CAS  PubMed  Google Scholar 

  52. Golovko MY, Murphy EJ (2008) Brain prostaglandin formation is increased by α-synuclein gene-ablation during global ischemia. Neurosci Lett 432:243–247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Golovko MY, Murphy EJ (2008) An improved LC–MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction. J Lipid Res 49:893–902

    Article  CAS  PubMed  Google Scholar 

  54. Anton RF, Wallis C, Randall CL (1983) In vivo regional levels of PGE and thromboxane in mouse brain: effect of decapitation, focused microwave fixation, and indomethacin. Prostaglandins 26:421–429

    Article  CAS  PubMed  Google Scholar 

  55. Farias SE, Basselin M, Chang L, Heidenreich KA, Rapoport SI, Murphy RC (2008) Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain. J Lipid Res 49:1990–2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Murphy EJ (2010) Brain fixation for analysis of brain lipid-mediators of signal transduction and brain eicosanoids requires head-focused microwave irradiation: an historical perspective. Prostaglandins Other Lipid Mediat 91:63–67

    Article  CAS  PubMed  Google Scholar 

  57. Galli C, Racagni G, Lands WEM, Smith WL (1982) Use of microwave techniques to inactivate brain enzymes rapidly. In: Colowick SP, Kaplan NO (eds) Methods enzymol. Academic Press, New York

    Google Scholar 

  58. Brose SA, Golovko MY (2013) Eicosanoid post-mortem induction in kidney tissue is prevented by microwave irradiation. Prostaglandins Leukot Essent Fatty Acids 89:313–318

    Article  CAS  PubMed  Google Scholar 

  59. Cenedella RJ, Galli C, Paoletti R (1975) Brain free fatty acid levels in rats sacrificed by decapitation versus focused microwave irradiation. Lipids 10:290–293

    Article  CAS  PubMed  Google Scholar 

  60. Kingsley PJ, Marnett LJ (2009) Analysis of endocannabinoids, their congeners and COX-2 metabolites. J Chromatogr B 877:2746–2754

    Article  CAS  Google Scholar 

  61. Bazinet RP, Lee H-J, Felder CC, Porter AC, Rapoport SI, Rosenberger TA (2005) Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain. Neurochem Res 30:597–601

    Article  CAS  PubMed  Google Scholar 

  62. Brose SA, Thuen BT, Golovko MY (2011) LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes. J Lipid Res 52:850–859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Raatz SK, Golovko MY, Brose SA, Rosenberger TA, Burr GS, Wolters WR, Picklo MJ (2011) Baking reduces prostaglandin, resolvin, and hydroxy-fatty acid content of farm-raised Atlantic Salmon (Salmo salar). J Agric Food Chem 59:11278–11286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Brose S, Baker A, Golovko M (2013) A fast one-step extraction and UPLC–MS/MS analysis for E2/D2 series prostaglandins and isoprostanes. Lipids 48:411–419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Golovko M, Barceló-Coblijn G, Castagnet P, Austin S, Combs C, Murphy E (2009) The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response. Mol Cell Biochem 326:55–66

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This publication was made possible by NIH Grant 5R01AG042819-04 (MG), NIH funded COBRE Mass Spec Core Facility Grant 5P30GM103329-04 (MG), and UND Office of the Vice President for Research Enhancement Award. We thank Ms. Amanda Marquardt for her excellent assistance with sample preparation.

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  1. Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, 501 N. Columbia Rd., Grand Forks, ND, 58202-9037, USA

    Stephen A. Brose, Svetlana A. Golovko & Mikhail Y. Golovko

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  1. Stephen A. Brose
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  2. Svetlana A. Golovko
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  3. Mikhail Y. Golovko
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Correspondence to Mikhail Y. Golovko.

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Brose, S.A., Golovko, S.A. & Golovko, M.Y. Brain 2-Arachidonoylglycerol Levels Are Dramatically and Rapidly Increased Under Acute Ischemia-Injury Which Is Prevented by Microwave Irradiation. Lipids 51, 487–495 (2016). https://doi.org/10.1007/s11745-016-4144-y

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