Metabolic Brain Disease

, Volume 28, Issue 3, pp 367–374

NeuroProtective effects of adenosine receptor agonist coadministration with ascorbic acid on CA1 hippocampus in a mouse model of ischemia reperfusion injury

Original Paper


Ischemic brain injury is a leading cause of sever neurological and neurobehavioral deficits and death. The hippocampus plays vital roles in learning and memory processes and it is impaired by ischemic insults. Cerebral ischemia/reperfusion leads to Oxidative stress damage impairing the hippocampus. Here we tested whether ascorbic acid and adenosine receptor played a neuroprotective role in a mouse brain ischemia model induced by common carotid arteries occlusion. Adult male mice were randomly assigned into nine experimental groups. The animals were subjected to ischemia by the ligation of common carotid arteries for 15 min. Drugs were injected intrapritoneally once daily for 7 days. Behavioral tests performed at day 14 and then mice were killed at day 21 and their brains were fixed for microscopic studies and some samples were prepared for western blot analysis. Western blot analysis utilized to evaluate the expression of apoptosis-related proteinsin the hippocampus. Short-term memory was assessed by shuttle-box test. Our findings revealed that administration of vitamin C and N6-cyclopentyladenosine (CPA) significantly attenuated ischemia-induced brain injury. Vitamin C and CPA administration increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of pro-apoptotic protein Bax in the ischemic mice. Ischemia caused short-term memory loss that was improved by vitamin c and CPA treatment. Our results demonstrate that treatment with vitamin C and adenosine receptor agonist attenuated cerebral ischemia/reperfusion-induced brain injury as a potential neuroprotective agent.


Hippocampus Neuroprotection Adenosine receptor Memory 


  1. Aly H, Abd-Rabboh L, El-Dib M, Nawwar F, Hassan H, Aaref M, Abdelrahman S, Elsayed A (2009) Ascorbic acid combined with ibuprofen in hypoxic ischemic encephalopathy: a randomized controlled trial. J Perinatol 29:438–443PubMedCrossRefGoogle Scholar
  2. Anderson CA, Arciniegas DB (2010) Cognitive sequelae of hypoxic-ischemic brain injury: a review. NeuroRehabilitation 26(1):47–63PubMedGoogle Scholar
  3. Bin J, Wang Q, Zhuo YY, Xu JP, Zhang HT (2012) Piperphentonamine (PPTA) attenuated cerebral ischemia-induced memory deficits via neuroprotection associated with anti-apoptotic activity. Metab Brain Dis 27:495–505PubMedCrossRefGoogle Scholar
  4. Bracchi-Ricard V, Brambilla R (2008) Astroglial nuclear factor-kB regulates learning and memory synaptic plasticity in female mice. J Neurochem 104:611–623PubMedGoogle Scholar
  5. Chan PH (2001) Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 21:2–14PubMedCrossRefGoogle Scholar
  6. Clemens JA, Stephenson DT, Smalstig EB, Dixon EP, Little SP (1997) Global ischemia activates nuclear factor-kappa B in forebrain neurons of rats. Stroke 28:1073–1080PubMedCrossRefGoogle Scholar
  7. Colbourne F, Sutherl G, Auer R (1999) Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia. J Neurosci 19:4200–4210PubMedGoogle Scholar
  8. Crack PJ, Taylor JM (2005) Reactive oxygen species and the modulation of stroke. Free Radic Biol Med 38:1433–1444PubMedCrossRefGoogle Scholar
  9. Cunha RA (2005) Neuroprotection by adenosine in the brain: from A1 receptor activation to A2A receptor blockade. Purinergic Signal 1:111–134PubMedCrossRefGoogle Scholar
  10. Dai QX, Wang LL, Chao J (2012) Effects of injecting adenosine A1 receptor agonist into baihui (GV20) on the cerebral cortex in ischemia/reperfusion injury model rats. Zhongguo Zhong Xi Yi Jie He Za Zhi 32:390–393PubMedGoogle Scholar
  11. Domenici MR, de Carolis AS, Sagratella S (1996) Block by N6-L-phenylisopropyl-adenosine of the electrophysiological and morphological correlates of hippocampal ischaemic injury in the gerbil. Br J Pharmacol 118:1551–1557PubMedCrossRefGoogle Scholar
  12. Fredholm BB (2007) Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 14:1315–1323PubMedCrossRefGoogle Scholar
  13. Grenz A, Homann D, Eltzschig HK (2011) Extracellular adenosine: a safety signal that dampens hypoxia-induced inflammation during ischemia. Antioxid Redox Signal 15:2221–2234PubMedCrossRefGoogle Scholar
  14. Harrison FE, May JM (2009) Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radic Biol Med 46:719–730PubMedCrossRefGoogle Scholar
  15. Hatano E, Bennett BL, Manning AM (2001) NF-kappa B stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Gastroenterology 120:1251–1262PubMedCrossRefGoogle Scholar
  16. Henry PT, Chandy MJ (1993) Ascorbic acid and focal cerebral ischaemia in a primate model. Acta Neurochir (Wien) 123:87–91CrossRefGoogle Scholar
  17. Heo JH, Han SW, Lee SK (2005) Free radicals as triggers of brain edema formation after stroke. Free Radic Biol Med 39:51–70PubMedCrossRefGoogle Scholar
  18. Heron M (2011) Deaths: leading causes for 2007. Natl Vital Stat Rep 59:1–95PubMedGoogle Scholar
  19. Héron A, Lekieffre D, Le Peillet E, Lasbennes F, Seylaz J, Plotkine M, Boulu RG (1994) Effects of an A1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia. Brain Res 641:217–224PubMedCrossRefGoogle Scholar
  20. Herrmann O, Baumann B, de Lorenzi R, Muhammad S, Zhang W, Kleesiek J, Malfertheiner M, Köhrmann M, Potrovita I, Maegele I, Beyer C, Burke JR, Hasan MT, Bujard H, Wirth T, Pasparakis M, Schwaninger M (2005) IKK mediates ischemia-induced neuronal death. Nat Med 11:1322–1329PubMedCrossRefGoogle Scholar
  21. Hoque N, Cook MA, Karmazyn M (2000) Inhibition of alpha(1)-adrenergic-mediated responses in rat ventricular myocytes by adenosine A(1) receptor activation: role of the K(ATP) channel. J Pharmacol Exp Ther 294:770–777PubMedGoogle Scholar
  22. Hu S, Dong H, Zhang H, Wang S, Hou L, Chen S, Zhang J, Xiong L (2012) Noninvasive limb remote ischemic preconditioning contributes neuroprotective effects via activation of adenosine A1 receptor and redox status after transient focal cerebral ischemia in rats. Brain Res 1459:81–90PubMedCrossRefGoogle Scholar
  23. Huang J, Agus DB, Winfree CJ, Kiss S, Mack WJ, McTaggart RA, Choudhri TF, Kim LJ, Mocco J, Pinsky DJ, Fox WD, Israel RJ, Boyd TA, Golde DW, Connolly ES Jr (2001) Dehydroascorbic acid, a blood–brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke. Proc Natl Acad Sci U S A 98:11720–11724PubMedCrossRefGoogle Scholar
  24. Johansson B, Halldner L, Dunwiddie TV, Masino SA, Poelchen W, Giménez-Llort L, Escorihuela RM, Fernández-Teruel A, Wiesenfeld-Hallin Z, Xu XJ, Hårdemark A, Betsholtz C, Herlenius E, Fredholm BB (2001) Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor. Proc Natl Acad Sci U S A 98:9407–9412PubMedCrossRefGoogle Scholar
  25. Kim J, Kim M, Song JH, Lee HT (2008) Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl 14:845–854PubMedCrossRefGoogle Scholar
  26. Kovalenko T, Osadchenko I, Nikonenko A, Lushnikova I, Voronin K, Nikonenko I, Muller D, Skibo G (2006) Ischemia-induced modifications in hippocampal CA1 stratum radiatum excitatory synapses. Hippocampus 16:814–825PubMedCrossRefGoogle Scholar
  27. Li Z, Pang L, Fang F, Zhang G, Zhang J, Xie M, Wang L (2012) Resveratrol attenuates brain damage in a rat model of focal cerebral ischemia via up-regulation of hippocampal Bcl-2. Brain Res 1450:116–124PubMedCrossRefGoogle Scholar
  28. Liu H, Lo CR, Czaja MJ (2002) NF-kappaB inhibition sensitizes hepatocytes to TNF-induced apoptosis through a sustained activation of JNK and c-Jun. Hepatology 35:772–778PubMedCrossRefGoogle Scholar
  29. Liu H, Zhang X, Du Y, Ji H, Li S, Li L, Xing Y, Zhang X, Dong L, Wang C, Zhao K, Ji Y, Cao X (2012) Leonurine protects brain injury by increased activities of UCP4, SOD, CAT and Bcl-2, decreased levels of MDA and Bax, and ameliorated ultrastructure of mitochondria in experimental stroke. Brain Res 1474:73–81PubMedCrossRefGoogle Scholar
  30. Mack WJ, Mocco J, Ducruet AF, Laufer I, King RG, Zhang Y, Guo W, Pinsky DJ, Connolly ES Jr (2006) A cerebroprotective dose of intravenous citrate/sorbitol-stabilized dehydroascorbic acid is correlated with increased cerebral ascorbic acid and inhibited lipid peroxidation after murine reperfused stroke. Neurosurgery 59:383–388PubMedCrossRefGoogle Scholar
  31. Meheroz H, Rabadi, Kristal B (2007) Effect of vitamin C supplementation on stroke recovery: a case–control study. Clin Interv Aging 2:147–151CrossRefGoogle Scholar
  32. Memet S (2006) NF-kB functions in the nervous system: from development to disease. Biochem Pharmacol 72:1180–1195PubMedCrossRefGoogle Scholar
  33. Ming Z, Fan YJ, Yang X, Lautt WW (2006) Synergistic protection by S-adenosylmethionine with vitamins C and E on liver injury induced by thioacetamide in rats. Free Radic Biol Med 40:617–624PubMedCrossRefGoogle Scholar
  34. Miura S, Ishida-Nakajima W, Ishida A, Kawamura M, Ohmura A, Oguma R, Sato Y, Takahashi T (2009) Ascorbic acid protects the newborn rat brain from hypoxic-ischemia. Brain Dev 31:307–317PubMedCrossRefGoogle Scholar
  35. Mohamed RA, Agha AM, Nassar NN (2012) SCH58261 the selective adenosine A(2A) receptor blocker modulates ischemia reperfusion injury following bilateral carotid occlusion: role of inflammatory mediators. Neurochem Res 37:538–547PubMedCrossRefGoogle Scholar
  36. Muir SW, Harrow C, Dawson J, Lees KR, Weir CJ, Sattar N, Walters MR (2008) Allopurinol use yields potentially beneficial effects on inflammatory indices in those with recent ischemic stroke: a randomized, double-blind, placebo-controlled trial. Stroke 39:3303–3307PubMedCrossRefGoogle Scholar
  37. Nakase T, Yoshioka S, Suzuki A (2011) Free radical scavenger, edaravone, reduces the lesion size of lacunar infarction in human brain ischemic stroke. BMC Neurol 11:39PubMedCrossRefGoogle Scholar
  38. Nayak H, Prentice M, Milton SL (2011) Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle. J Cereb Blood Flow Metab 31:467–475PubMedCrossRefGoogle Scholar
  39. Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, Koistinaho J (2004) Nuclear factor-κB contributes to infarction after permanent focal ischemia. Stroke 35:987–991PubMedCrossRefGoogle Scholar
  40. Okoro C, Balluz L, Campbell V, Holt J, Mokdad A (2005) State and metropolitan-area estimates of disability in the United States. Am J Public Health 95:1964–1969PubMedCrossRefGoogle Scholar
  41. Park SW, Kim JY, Ham A, Brown KM, Kim M, D’Agati VD, Lee HT (2012) A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 303:F721–F732PubMedCrossRefGoogle Scholar
  42. Pearson T, Damian K, Lynas RE, Frenguelli BG (2006) Sustained elevation of extracellular adenosine and activation of A1 receptors underlie the post-ischaemic inhibition of neuronal function in rat hippocampus in vitro. J Neurochem 97:1357–1368PubMedCrossRefGoogle Scholar
  43. Pizzi M, Sarnico I, Boroni F, Benarese M, Steimberg N, Mazzoleni G, Dietz GP, Bähr M, Liou HC, Spano PF (2005) NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity. Cell Death Diff 12:761–772CrossRefGoogle Scholar
  44. Polidori MC, Praticó D, Ingegni T, Mariani E, Spazzafumo L, Del Sindaco P, Cecchetti R, Yao Y, Ricci S, Cherubini A, Stahl W, Sies H, Senin U, Mecocci P, AVASAS Study Group (2005) Effects of vitamin C and aspirin in ischemic stroke-related lipid peroxidation: results of the AVASAS (Aspirin Versus Ascorbic acid plus Aspirin in Stroke) Study. Biofactors 24:265–274PubMedCrossRefGoogle Scholar
  45. Ren J, Fan C, Chen N, Huang J, Yang Q (2011) Resveratrol pretreatment attenuates cerebral ischemic injury by upregulating expression of transcription factor Nrf2 and HO-1 in rats. Neurochem Res 36:2352–2362PubMedCrossRefGoogle Scholar
  46. Rudolphi KA, Schubert P, Parkinson FE, Fredholm BB (1992) Neuroprotective role of adenosine in cerebral ischaemia. Trends Pharmacol Sci 13:439–445PubMedCrossRefGoogle Scholar
  47. Saransaari P, Oja SS (2002) Mechanisms of adenosine release in the developing and adult mouse hippocampus. Neurochem Res 27:911–918PubMedCrossRefGoogle Scholar
  48. Schneider A, Martin-Villalba A, Weih F, Vogel J, Wirth T, Schwaninger M (1999) NF-kappaB is activated and promotes cell death in focal cerebral ischemia. Nat Med 5:554–559PubMedCrossRefGoogle Scholar
  49. Seta KA, Yuan Y, Spicer Z, Lu G, Bedard J, Ferguson TK, Pathrose P, Cole-Strauss A, Kaufhold A, Millhorn DE (2004) The role of calcium in hypoxia-induced signal transduction and gene expression. Cell Calcium 36:331–340PubMedCrossRefGoogle Scholar
  50. Shen H, Zhang L, Yuen D, Logan R, Jung BP, Zhang G, Eubanks JH (2002) Expression and function of A1 adenosine receptors in the rat hippocampus following transient forebrain ischemia. Neuroscience 114:547–556PubMedCrossRefGoogle Scholar
  51. Sheridan GK, Pickering M, Twomey CJ (2007) NF-kB activity in distinct neural subtypes of the rat hippocampus: influence of time and GABA antagonism in acute slice preparations. Learn Mem 14:525–532PubMedCrossRefGoogle Scholar
  52. Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM, Diener HC, Ashwood T, Wasiewski WW, Emeribe U, SAINT II Trial Investigators (2006) NXY-059 for the treatment of acute ischemic stroke. N Engl J Med 354:588–600PubMedCrossRefGoogle Scholar
  53. von der Leyen H, Schmitz W, Scholz H, Scholz J, Lohse MJ, Schwabe U (1989) Effects of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly selective adenosine receptor antagonist, on force of contraction in guinea-pig atrial and ventricular cardiac preparations. Naunyn Schmiedebergs Arch Pharmacol 340:204–209PubMedCrossRefGoogle Scholar
  54. Yoshida M, Watanabe C, Satoh M, Yasutake A, Sawada M, Ohtsuka Y, Akama Y, Tohyama C (2004) Susceptibility of metallothionein-null mice to the behavioural alterations caused by exposure to mercury vapor at human-relevant concentration. Toxicol Sci 80:69–73Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Cellular and Molecular Research Center and Department of AnatomyTehran University of Medical SciencesTehranIran
  2. 2.Research Center for Stem Cell TherapyHormozgan University of Medical SciencesBandar AbbasIran

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