Neurochemical Research

, Volume 37, Issue 4, pp 749–758 | Cite as

Preconditioning with a Novel Metallopharmaceutical NO Donor in Anesthetized Rats Subjected to Brain Ischemia/Reperfusion

  • Marcio Wilker Soares Campelo
  • Reinaldo Barreto Oriá
  • Luiz Gonzaga de França Lopes
  • Gerly Anne de Castro Brito
  • Armenio Aguiar dos Santos
  • Raquel Cavalcante de Vasconcelos
  • Francisco Ordelei Nascimento da Silva
  • Beatrice Nuto Nobrega
  • Moisés Tolentino Bento-Silva
  • Paulo Roberto Leitão de Vasconcelos
Original Paper


Rut-bpy is a novel nitrosyl–ruthenium complex releasing NO into the vascular system. We evaluated the effect of Rut-bpy (100 mg/kg) on a rat model of brain stroke. Forty rats were assigned to four groups (Saline solution [SS], Rut-bpy, SS+ischemia–reperfusion [SS+I/R] and Rut-bpy+ischemia–reperfusion [Rut-bpy+I/R]) with their mean arterial pressure (MAP) continuously monitored. The groups were submitted (SS+I/R and Rut-bpy+I/R) or not (SS and Rut-bpy) to incomplete global brain ischemia by occlusion of the common bilateral carotid arteries during 30 min followed by reperfusion for further 60 min. Thirty minutes before ischemia, rats were treated pairwise by intraperitoneal injection of saline solution or Rut-bpy. At the end of experiments, brain was removed for triphenyltetrazolium chloride staining in order to quantify the total ischemic area. In a subset of rats, hippocampus was obtained for histopathology scoring, nitrate and nitrite measurements, immunostaining and western blotting of the nuclear factor- κB (NF-κB). Rut-bpy pre-treatment decreased MAP variations during the transition from brain ischemia to reperfusion and decreased the fractional injury area. Rut-bpy pre-treatment reduced NF-κB hippocampal immunostaining and protein expression with improved histopathology scoring as compared to the untreated operated control. In conclusion, Rut-bpy improved the total brain infarction area and hippocampal neuronal viability in part by inhibiting NF-κB signaling and helped to stabilize the blood pressure during the transition from ischemia to reperfusion.


Brain Ischemia–reperfusion Nitric oxide Nitrosyl–ruthenium complex Nuclear factor-κB 



We are in debt to PhD. Prof. Renata F. C. Leitão, Rossângela Barreto, Antônio Haroldo Pinheiro Ferreira, and Maria Silvandira França Pinheiro for their helpful technical assistance. CNPq and FUNCAP scholarships and research grants supported this work.

Conflict of interest

There are no actual or potential conflicts of interests.


  1. 1.
    Kung HC, Hoyert DL, Xu J, Murphy SL (2008) Deaths: final data for 2005. National Vital Statistics Reports 56:1–120Google Scholar
  2. 2.
    Dawson VL, Dawson TM (1998) Nitric oxide in neurodegeneration. Prog Brain Res 118:215–229PubMedCrossRefGoogle Scholar
  3. 3.
    Dawson VL, Dawson TM (1995) Physiological and toxicological actions of nitric oxide in the central nervous system. Adv Pharmacol 34:323–342PubMedCrossRefGoogle Scholar
  4. 4.
    Willmot M, Gibson C, Gray L, Murphy S, Bath P (2005) Nitric oxide synthase inhibitors in experimental ischemic stroke and their effects on infarct size and cerebral blood flow: a systematic review. Free Radic Biol Med 39:412–425PubMedCrossRefGoogle Scholar
  5. 5.
    Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84:9265–9269PubMedCrossRefGoogle Scholar
  6. 6.
    Dimmeler S, Haendeler J, Nehls M, Zeiher AM (1997) Suppression of apoptosis by nitric oxide via inhibition of interleukin-1 b-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med 185:601–607PubMedCrossRefGoogle Scholar
  7. 7.
    Rossig L, Haendeler J, Hermann C et al (2000) Nitric oxide down-regulates MKP-3 mRNA levels: involvement in endothelial cell protection from apoptosis. J Biol Chem 275:25502–25507PubMedCrossRefGoogle Scholar
  8. 8.
    Zhuang P, Ji H, Zhang YH, Min ZL, Ni QG, You R (2010) ZJM-289, a Novel nitric oxide donor, alleviates the cerebral ischemic–reperfusion injury in rats. Clin Exp Pharmacol Physiol 37:e121–e127PubMedCrossRefGoogle Scholar
  9. 9.
    Ignarro LJ, Cirino G, Casini A, Napoli C (1999) Nitric oxide as a signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol 34:879–886PubMedCrossRefGoogle Scholar
  10. 10.
    Ignarro LJ, Napoli C, Loscalzo J (2002) Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res 90:21–28PubMedCrossRefGoogle Scholar
  11. 11.
    Wainwright MS, Grundhoefer D, Sharma S, Black SM (2007) A nitric oxide donor reduces brain injury and enhances recovery of cerebral blood flow after hypoxia-ischemia in the newborn rat. Neurosci Lett 415:124–129PubMedCrossRefGoogle Scholar
  12. 12.
    Khan M, Jatana M, Elango C, Paintlia AS, Singh AK, Singh I (2006) Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke. Nitric Oxide 15:114–124PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang R, Zhang L, Zhang Z, Wang Y, Lu M, Lapointe M, Chopp M (2001) A nitric oxide donor induces neurogenesis and reduces functional deficits after stroke in rats. Ann Neurol 50:602–611PubMedCrossRefGoogle Scholar
  14. 14.
    Silva FON, Araujo SXB, Holanda AKM, Meyer E, Sales FAM, Diogenes ICN, Carvalho IMM, Moreira IS, Lopes LGF (2006) Synthesis, Characterization, and NO Release Study of the cis- and trans-[Ru(Bpy)2(SO3)(NO)]+ complexes Eur J Inorg Chem 2020–2026Google Scholar
  15. 15.
    Fricker SP, Slade E, Powell NA, Vaughan OJ, Henderson GR, Murrer BA et al (1997) Ruthenium complexes as nitric oxide scavengers: a potential therapeutic approach to nitric oxide-mediated diseases. Br J Pharmacol 122:1441–1449PubMedCrossRefGoogle Scholar
  16. 16.
    Hutchings SR, Song D, Fricker SP, Pang CC (2005) The ruthenium-based nitric oxide scavenger, AMD6221, augments cardiovascular responsiveness to noradrenaline in rats with streptozotocin-induced diabetes. Eur J Pharmacol 528:132–136PubMedCrossRefGoogle Scholar
  17. 17.
    Silva JJN, Guedes PMM, Zottis A, Balliano TL, Silva FON, Lopes LGF, Ellena J, Oliva G, Andricopulo AD, Franco DW, Silva JS (2010) Novel ruthenium complexes as potential drugs for Chagas’s disease: enzyme inhibition and in vitro/in vivo trypanocidal activity. Br J Pharmacol 160:260–269PubMedCrossRefGoogle Scholar
  18. 18.
    Cerqueira JB, Silva LF, Lopes LG, Moraes ME, Nascimento NR (2008) Relaxation of rabbit corpus cavernosum smooth muscle and aortic vascular endothelium induced by new nitric oxide donor substances of the nitrosyl–ruthenium complex. Int Braz J Urol 34:638–646PubMedCrossRefGoogle Scholar
  19. 19.
    Silva FO, Cândido MC, Holanda AK, Diógenes IC, Sousa EH, Lopes LG (2011) Mechanism and biological implications of the NO release of cis-[Ru(bpy)2L(NO)]n+ complexes: a key role of physiological thiols. J Inorg Biochem 105:624–629PubMedCrossRefGoogle Scholar
  20. 20.
    Bonaventura D, de Lima RG, Vercesi JA, da Silva RS, Bendhack LM (2007) Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: sodium nitroprusside and a new ruthenium complex. Vascul Pharmaco 46:215–222CrossRefGoogle Scholar
  21. 21.
    Muniz LRF, Faria MHG, Vasconcelos PRL (2004) Metabolic evaluation of ischemic and reperfusion brain injury following bilateral occlusion of common carotid arteries: an experimental study in rats. Acta Cir Bras 19:529–534CrossRefGoogle Scholar
  22. 22.
    Ibayashi S, Nagao T, Kitazono T, Ooboshi H, Kitayama J, Sadoshima S, Fujishima M (2000) Calcium antagonist is radipine reduces metabolic alterations in acute cerebral ischemia in spontaneously hypertensive rats. Neurochem Res 25:349–355PubMedCrossRefGoogle Scholar
  23. 23.
    Li Z, Wang Y, Xie Y, Yang Z, Zhang T (2011) Protective effects of exogenous hydrogen sulfide on neurons of hippocampus in a rat model of brain ischemia. Neurochem Res 36:1840–1849PubMedCrossRefGoogle Scholar
  24. 24.
    Paxinos G, Watson C (2004) The rat brain in stereotaxic coordinates, 5th edn. Elsevier Academic Press, BurlingtonGoogle Scholar
  25. 25.
    Joshi CN, Jain SK, Murthy PS (2004) An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts. Brain Res Brain Res Protoc 13:11–17PubMedCrossRefGoogle Scholar
  26. 26.
    Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM (1986) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304–1308PubMedCrossRefGoogle Scholar
  27. 27.
    Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing LG Jr et al (2006) Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res 1116:159–165PubMedCrossRefGoogle Scholar
  28. 28.
    Isayama K, Pitts LH, Nishimura MC (1991) Evaluation of 2, 3, 5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke 22:1394–1398PubMedCrossRefGoogle Scholar
  29. 29.
    Leinonen JS, Ahonen JP, Lonnrot K, Jehkonen M, Dastidar P, Molnar G, Alho H (2000) Low plasma antioxidant activity is associated with high lesion volume and neurological impairment in stroke. Stroke 31:33–39PubMedCrossRefGoogle Scholar
  30. 30.
    Goldlust EJ, Paczynski RP, He YY, Hsu CY, Goldberg MP (1996) Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride-stained rat brains. Stroke 27:1657–1662PubMedCrossRefGoogle Scholar
  31. 31.
    Kaku Y, Yonekawa Y, Tsukahara T, Ogata N, Kimura T, Taniguchi T (1993) Alterations of a 200 kDa Neurofilament in the Rat Hippocampus after Forebrain Ischemia. J Cereb Blood Flow Metab 13:402–408PubMedCrossRefGoogle Scholar
  32. 32.
    Green LC, Wagner DA, Glogowski J (1982) Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126:131–138PubMedCrossRefGoogle Scholar
  33. 33.
    Irmak MK, Fadillioglu E, Sogut S, Erdogan H, Gulec M, Ozer M, Yagmurca M, Gozukara ME (2003) Effects of caffeic acid phenethyl ester and alpha-tocopherol on reperfusion injury in rat brain. Cell Biochem Funct 21:283–289PubMedCrossRefGoogle Scholar
  34. 34.
    Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391–397PubMedCrossRefGoogle Scholar
  35. 35.
    Lo EH, Moskowitz MA, Jacobs TP (2005) Exciting, radical, suicidal: how brain cells die after stroke. Stroke 36:189–192PubMedCrossRefGoogle Scholar
  36. 36.
    Prieto-Arribas R, Pascual-Garvi JM, González-Llanos F, Roda JM (2011) How to repair an ischemic brain injury? Value of experimental models in search of answers. Neurologia 26:65–73PubMedGoogle Scholar
  37. 37.
    Pluta RM, Rak R, Wink DA, Woodward JJ, Khaldi A, Oldfield EH, Watson JC (2001) Effects of nitric oxide on reactive oxygen species production and infarction size after brain reperfusion injury. Neurosurgery 48:884–892PubMedGoogle Scholar
  38. 38.
    Zhang D, Dhillon HS, Mattson MP, Yurek DM, Prasad RM (1999) Immunohistochemical detection of the lipid peroxidation product 4-hydroxynonenal after experimental brain injury in the rat. Neurosci Lett 272:57–61PubMedCrossRefGoogle Scholar
  39. 39.
    Gaur V, Kumar A (2010) Protective effect of desipramine, venlafaxine and trazodone against experimental animal model of transient global ischemia: possible involvement of NO-cGMP pathway. Brain Res 1353:204–212PubMedCrossRefGoogle Scholar
  40. 40.
    Buisson A, Plotkine M, Boulu RG (1992) The neuroprotective effect of a nitric oxide inhibitor in a rat model of focal cerebral ischaemia. Br J Pharmacol 106:766–767PubMedGoogle Scholar
  41. 41.
    Lei H, Grinberg O, Nwaigwe CI, Hou HG, Williams H, Swartz HM, Dunn JF (2001) The effects of ketamine–xylazine anesthesia on cerebral blood flow and oxygenation observed using nuclear magnetic resonance perfusion imaging and electron paramagnetic resonance oximetry. Brain Res 913:174–179PubMedCrossRefGoogle Scholar
  42. 42.
    Stein AB, Tiwari S, Thomas P, Hunt G, Levent C, Stoddard MF, Tang XL, Bolli R, Dawn B (2007) Effects of anesthesia on echocardiographic assessment of left ventricular structure and function in rats. Basic Res Cardiol 102:28–41PubMedCrossRefGoogle Scholar
  43. 43.
    Levine S (1960) Anoxic–ischemic encephalopathy in rats. Am J Pathol 36:1–17PubMedGoogle Scholar
  44. 44.
    Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431–1568PubMedGoogle Scholar
  45. 45.
    He Z, Ibayashi S, Sugimori H, Fujii K, Sadoshima S, Fujishima M (1997) Age-related ischemia in the brain following bilateral carotid artery occlusion—collateral blood flow and brain metabolism. Neurochem Res 22:37–42PubMedCrossRefGoogle Scholar
  46. 46.
    Glenner GG (1969) Tetrazolium salts. In: Lillie RD (ed) H.J. Con’s biological stains. Williams and Wilkins, Baltimore, pp 154–162Google Scholar
  47. 47.
    Li F, Irie K, Anwer MS, Fisher M (1997) Delayed triphenyltetrazolium chloride staining remains useful for evaluating cerebral infarct volume in a rat stroke model. J Cereb Blood Flow Metab 17:1132–1135PubMedCrossRefGoogle Scholar
  48. 48.
    Marshall RS, Lazar RM, Pile-Spellman J, Young WL, Duong DH, Joshi S, Ostapkovich N (2001) Recovery of brain function during induced cerebral hypoperfusion. Brain 124:1208–1217PubMedCrossRefGoogle Scholar
  49. 49.
    Willmot MR, Bath PM (2003) The potential of nitric oxide therapeutics in stroke. Expert Opin Investig Drugs 12:455–470PubMedCrossRefGoogle Scholar
  50. 50.
    Sanchez A, Fernandez N, Monge L, Salcedo A, Climent B, Luis Garcia-Villalon A, Diéquez G (2006) Goat cerebrovascular reactivity to ADP after ischemia–reperfusion. Role of nitric oxide, prostanoids and reactive oxygen species. Brain Res 1120:114–123PubMedCrossRefGoogle Scholar
  51. 51.
    Wieraszkoa A, Clarkeb MJ, Langb DR, Lopes LGF, Franco DW (2001) The influence of NO-containing ruthenium complexes on mouse hippocampal evoked potentials in vitro. Life Sci 68:1535–1544CrossRefGoogle Scholar
  52. 52.
    Tardini DMS, Winston BY, Novelli ELB, Sequeira JL (2003) Evaluation of two brain ischemia and reperfusion experimental models in rats with carotid temporary occlusion associated or not to vertebral occlusion. Acta Cir Bras 18:514–517CrossRefGoogle Scholar
  53. 53.
    De La Torre JC, Aliev G (2005) Inhibition of vascular nitric oxide after rat chronic brain hypoperfusion: spatial memory and immunocytochemical changes. J Cereb Blood Flow Metab 25:663–672PubMedCrossRefGoogle Scholar
  54. 54.
    Greco R, Mangione AS, Amantea D, Bagetta G, Nappi G, Tassorelli C (2011) IkappaB-alpha expression following transient focal cerebral ischemia is modulated by nitric oxide. Brain Res 1372:145–151PubMedCrossRefGoogle Scholar
  55. 55.
    Kaltschmidt B, Heinrich M, Kaltschmidt C (2002) Stimulus-dependent activation of NF-kappaB specifies apoptosis or neuroprotection in cerebellar granule cells. Neuromolecular Med 2:299–309PubMedCrossRefGoogle Scholar
  56. 56.
    Medling BD, Bueno R, Chambers C, Neumeister MW (2010) The effect of vitamin E succinate on ischemia reperfusion injury. Hand 5:60–64CrossRefGoogle Scholar
  57. 57.
    Wang Q, Tang XN, Yenari MA (2007) The inflammatory response in stroke. J Neuroimmunol 184:53–68PubMedCrossRefGoogle Scholar
  58. 58.
    Desai A, Singh N, Raghubir R (2010) Neuroprotective potential of the NF-κB inhibitor peptide IKK-NBD in cerebralischemia–reperfusion injury. Neurochem Int 57:876–883PubMedCrossRefGoogle Scholar
  59. 59.
    Lefer AM, Lefer DJ (1993) Pharmacology of the endothelium in ischemia–reperfusion and circulatory shock. Annu Rev Pharmacol Toxicol 33:71–90PubMedCrossRefGoogle Scholar
  60. 60.
    Thiagarajan RR, Winn RK, Harlan JM (1997) The role of leukocyte and endothelial adhesion molecules in ischemia–reperfusion injury. Thromb Haemost 78:310–314PubMedGoogle Scholar
  61. 61.
    Goto S, Xue R, Sugo N, Sawada M, Blizzard KK, Poitras MF, Johns DC, Dawson TM, Dawson VL, Crain BJ, Traystman RJ, Mori S, Hurn PD (2002) Poly(ADP-ribose) polymerase impairs early and long-term experimental stroke recovery. Stroke 33:1101–1106PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Marcio Wilker Soares Campelo
    • 1
  • Reinaldo Barreto Oriá
    • 2
  • Luiz Gonzaga de França Lopes
    • 3
  • Gerly Anne de Castro Brito
    • 2
  • Armenio Aguiar dos Santos
    • 4
  • Raquel Cavalcante de Vasconcelos
    • 5
  • Francisco Ordelei Nascimento da Silva
    • 3
  • Beatrice Nuto Nobrega
    • 5
  • Moisés Tolentino Bento-Silva
    • 2
  • Paulo Roberto Leitão de Vasconcelos
    • 1
  1. 1.Department of SurgeryFederal University of CearáFortalezaBrazil
  2. 2.Department of MorphologyFederal University of CearáFortalezaBrazil
  3. 3.Department of Organic and Inorganic ChemistryFederal University of CearáFortalezaBrazil
  4. 4.Department of Physiology and PharmacologyFederal University of CearáFortalezaBrazil
  5. 5.Student of the Medical SchoolFederal University of CearáFortalezaBrazil

Personalised recommendations