Neuroprotective effect of diazoxide on brain injury induced by cerebral ischemia/reperfusion during deep hypothermia



      The purpose of this study was to determine the effects of diazoxide on apoptosis and the relative mechanisms in a model of brain injury induced by cerebral ischemia/reperfusion (I/R) during deep hypothermia.


      Three-week-old Sprague–Dawley male rats were randomly and equitably divided into sham-operated group, placebo-treated group and diazoxide-treated group respectively. Specific examination of the regional cerebral blood flow (rCBF) was measured in the three groups continuously during the operation by laser Doppler flowmetry. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was showed DNA fragmentation. The mRNA expressions of cytochrome c and full-length caspase-3 were determined by RT-PCR, while the protein expressions of cytochrome c and cleaved caspase-3 were determined by immunohistochemistry at 1 h, 6 h, 24 h, 72 h and 7 days after I/R, respectively. Cytosolic release of cytochrome c at 24 h after I/R was also confirmed by Western blot.


      rCBF was significantly decreased in both of placebo-treated and diazoxide-treated group just after ischemia in the time interval 0–5 min, and had no obvious changes in all the time intervals during the operation. Diazoxide preconditioning significantly decreased the percentage of TUNEL-positive staining cells. The mRNA expressions of cytochrome c and full-length caspase-3 in diazoxide-treated group were significantly decreased. In addition, diazoxide provided a significant reduction in the protein expressions of cytochrome c and cleaved caspase-3.


      These results suggested that the neuroprotective effects of diazoxide against cerebral I/R injury during deep hypothermia correlated with the reduction of DNA fragmentation, prevention of mitochondrial cytochrome c release and inhibition of caspase-3 activation.


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        • Kerendi F.
        • Halkos M.E.
        • Kin H.
        • Corvera J.S.
        • Brat D.J.
        • Wagner M.B.
        • et al.
        Upregulation of hypoxia inducible factor is associated with attenuation of neuronal injury in neonatal piglets undergoing deep hypothermic circulatory arrest.
        J Thorac Cardiovasc Surg. 2005; 130: 1079-1089
        • Kinney H.C.
        • Panigrahy A.
        • Newburger J.W.
        • Jonas R.A.
        • Sleeper L.A.
        Hypoxic-ischemic brain injury in infants with congenital heart disease dying after cardiac surgery.
        Acta Neuropathol (Berl). 2005; 110: 563-578
        • Clancy R.R.
        • McGaurn S.A.
        • Wernovsky G.
        • Gaynor J.W.
        • Spray T.L.
        • Norwood W.I.
        • et al.
        Risk of seizures in survivors of newborn heart surgery using deep hypothermic circulatory arrest.
        Pediatrics. 2003; 111: 592-601
        • Liu D.
        • Lu C.
        • Wan R.
        • Auyeung W.W.
        • Mattson M.P.
        Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release.
        J Cereb Blood Flow Metab. 2002; 22: 431-443
        • Shimizu K.
        • Lacza Z.
        • Rajapakse N.
        • Horiguchi T.
        • Snipes J.
        • Busija D.W.
        MitoK(ATP) opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat.
        Am J Physiol Heart Circ Physiol. 2002; 283: 1005-1011
        • Domoki F.
        • Kis B.
        • Naqy K.
        • Farkas E.
        • Busija D.W.
        • Bari F.
        Diazoxide preserves hypercapnia-induced arteriolar vasodilation after global cerebral ischemia in piglets.
        Am J Physiol Heart Circ Physiol. 2005; 289: 368-373
        • Kis B.
        • Rajapakse N.C.
        • Snipes J.A.
        • Nagy K.
        • Horiguchi T.
        • Busija D.W.
        Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons.
        J Neurochem. 2003; 87: 969-980
        • Nakaqawa I.
        • Alessandri B.
        • Heimann A.
        • Kempski O.
        MitoKATP-channel opener protects against neuronal death in rat venous ischemia.
        Neurosurgery. 2005; 57: 334-340
        • Kowaltowski A.J.
        • Maciel E.N.
        • Fornazari M.
        • Castilho R.F.
        Diazoxide protects against methylmalonate-induced neuronal toxicity.
        Exp Neurol. 2006; 201: 165-171
        • Hausenloy D.J.
        • Yellon D.M.
        • Mani-Babu S.
        • Duchen M.R.
        Preconditioning protects by inhibiting the mitochondrial permeability transition.
        Am J Physiol Heart Circ Physiol. 2004; 287: 841-849
        • Wu L.
        • Shen F.
        • Lin L.
        • Zhang X.
        • Bruce I.C.
        • Xia Q.
        The neuroprotection conferred by activating the mitochondrial ATP-sensitive K+ channel is mediated by inhibiting the mitochondrial permeability transition pore.
        Neurosci Lett. 2006; 402: 184-189
        • Busija D.W.
        • Katakam P.
        • Rajapakse N.C.
        • Kis B.
        • Grover G.
        • Domoki F.
        • et al.
        Effects of ATP-sensitive potassium channel activators diazoxide and BMS-191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria.
        Brain Res Bull. 2005; 66: 85-90
        • Hoyte L.C.
        • Papadakis M.
        • Barber P.A.
        • Buchan A.M.
        Improved regional cerebral blood flow is important for the protection seen in a mouse model of late phase ischemic preconditioning.
        Brain Res. 2006; 1121: 231-237
        • Wang Y.C.
        • Lin C.W.
        • Shen C.C.
        • Lai S.C.
        • Kuo J.S.
        Tissue plasminogen activator for the treatment of intraventricular hematoma: the dose-effect relationship.
        J Neurol Sci. 2002; 202: 35-41
        • Kim C.S.
        • Park J.B.
        • Kim K.J.
        • Chang S.J.
        • Ryoo S.W.
        • Jeon B.H.
        Effect of Korea red ginseng on cerebral blood flow and superoxide production.
        Acta Pharmacol Sin. 2002; 23: 1152-1156
        • Ohmura A.
        • Nakajima W.
        • Ishida A.
        • Yasuoka N.
        • Kawamura M.
        • Miura S.
        • et al.
        Prolonged hypothermia protects neonatal rat brain against hypoxic-ischemia by reducing both apoptosis and necrosis.
        Brain Dev. 2005; 7: 517-526
        • Bagetta G.
        • Chiappetta O.
        • Amantea D.
        • Iannone M.
        • Rotiroti D.
        • Costa A.
        • et al.
        Estradiol reduces cytochrome c translocation and minimizes hippocampal damage caused by transient global ischemia in rat.
        Neurosci Lett. 2004; 368: 87-91
        • Niquet J.
        • Seo D.W.
        • Wasterlain C.G.
        Mitochondrial pathways of neuronal necrosis.
        Biochem Soc Trans. 2006; 34: 1347-1351
        • Ito Y.
        • Oh-Hashi K.
        • Kiuchi K.
        • Hirata Y.
        p44/42 MAP kinase and c-Jun N-terminal kinase contribute to the up-regulation of caspase-3 in manganese-induced apoptosis in PC12 cells.
        Brain Res. 2006; 1099: 1-7
        • McCullough J.N.
        • Zhang N.
        • Reich D.L.
        • Juvonen T.S.
        • Klein J.J.
        • Spielvogel D.
        • et al.
        Cerebral metabolic suppression during hypothermic circulatory arrest in humans.
        Ann Thorac Surg. 1999; 67 (discussion 1919–1921): 1895-1899
        • Ehrlich M.P.
        • McCullough J.N.
        • Zhang N.
        • Weisz D.J.
        • Juvonen T.
        • Bodian C.A.
        • et al.
        Effect of hypothermia on cerebral blood flow and metabolism in the pig.
        Ann Thorac Surg. 2002; 73: 191-197
        • Panos A.
        • Murith N.
        • Bednarkiewicz M.
        • Khatchatourov G.
        Axillary cerebral perfusion for arch surgery in acute type A dissection under moderate hypothermia.
        Eur J Cardiothorac Surg. 2006; 29: 1036-1039
        • Ananiadou O.G.
        • Bibou K.
        • Drossos G.E.
        • Charchanti A.
        • Bai M.
        • Haj-Yahia S.
        • et al.
        Effect of profound hypothermia during circulatory arrest on neurologic injury and apoptotic repressor protein Bcl-2 expression in an acute porcine model.
        J Thorac Cardiovasc Surg. 2007; 133: 919-926
        • Ditsworth D.
        • Priestley M.A.
        • Loepke A.W.
        • Ramamoorthy C.
        • McCann J.
        • Staple L.
        • et al.
        Apoptotic neuronal death following deep hypothermic circulatory arrest in piglets.
        Anesthesiology. 2003; 98: 1119-1127
        • Scallan M.J.
        Cerebral injury during paediatric heart surgery: perfusion issues.
        Perfusion. 2004; 19: 221-228
        • Amir G.
        • Ramamoorthy C.
        • Riemer R.K.
        • Reddy V.M.
        • Hanley F.L.
        Neonatal brain protection and deep hypothermic circulatory arrest: pathophysiology of ischemic neuronal injury and protective strategies.
        Ann Thorac Surg. 2005; 80: 1955-1964
        • Gaynor J.W.
        • Nicolson S.C.
        • Jarvik G.P.
        • Wernovsky G.
        • Montenegro L.M.
        • Burnham N.B.
        • et al.
        Increasing duration of deep hypothermic circulatory arrest is associated with an increased incidence of postoperative electroencephalographic seizures.
        J Thorac Cardiovasc Surg. 2005; 130: 1278-1286
        • Gaynor J.W.
        • Wernovsky G.
        • Jarvik G.P.
        • Bernbaum J.
        • Gerdes M.
        • Zackai E.
        • et al.
        Patient characteristics are important determinants of neurodevelopmental outcome at one year of age after neonatal and infant cardiac surgery.
        J Thorac Cardiovasc Surg. 2007; 133: 1344-1353
        • Sheng H.
        • Laskowitz D.T.
        • Pearlstein R.D.
        • Warner D.S.
        Characterization of a recovery global cerebral ischemia model in the mouse.
        J Neurosci Methods. 1999; 88: 103-109
        • Muller G.J.
        • Stadelmann C.
        • Bastholm L.
        • Elling F.
        • Lassmann H.
        • Johansen F.F.
        Ischemia leads to apoptosis- and necrosis-like neuron death in the ischemic rat hippocampus.
        Brain Pathol. 2004; 14: 415-424
        • Wei L.
        • Ying D.J.
        • Cui L.
        • Langsdorf J.
        • Yu S.P.
        Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats.
        Brain Res. 2004; 1022: 54-61
        • Tanaka H.
        • Yokota H.
        • Jover T.
        • Cappuccio I.
        • Calderone A.
        • Simionescu M.
        • et al.
        Ischemic preconditioning: neuronal survival in the face of caspase-3 activation.
        J Neurosci. 2004; 24: 2750-2759
        • Lau A.
        • Arundine M.
        • Sun H.S.
        • Jones M.
        • Tymianski M.
        Inhibition of caspase-mediated apoptosis by peroxynitrite in traumatic brain injury.
        J Neurosci. 2006; 26: 11540-11553
        • Tsubokawa T.
        • Jadhav V.
        • Solaroglu I.
        • Shiokawa Y.
        • Konishi Y.
        • Zhang J.H.
        Lecithinized superoxide dismutase improves outcomes and attenuates focal cerebral ischemic injury via antiapoptotic mechanisms in rats.
        Stroke. 2007; 38: 1057-1062
        • Nijboer C.H.
        • Groenendaal F.
        • Kavelaars A.
        • Hagberg H.H.
        • van Bel F.
        • Heijnen C.J.
        Gender-specific neuroprotection by 2-iminobiotin after hypoxia-ischemia in the neonatal rat via a nitric oxide independent pathway.
        J Cereb Blood Flow Metab. 2007; 27: 282-292
        • Sugawara T.
        • Noshita N.
        • Lewen A.
        • Gasche Y.
        • Ferrand-Drake M.
        • Fujimura M.
        • et al.
        Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation.
        J Neurosci. 2002; 22: 209-217
        • Noto T.
        • Ishiye M.
        • Furuich Y.
        • Keida Y.
        • Katsuta K.
        • Moriguchi A.
        • et al.
        Neuroprotective effect of tacrolimus (FK506) on ischemic brain damage following permanent focal cerebral ischemia in the rat.
        Brain Res Mol Brain Res. 2004; 128: 30-38
        • Twiddy D.
        • Brown D.G.
        • Adrain C.
        • Jukes R.
        • Martin S.J.
        • Cohen G.M.
        • et al.
        Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex.
        J Biol Chem. 2004; 279: 19665-19682
        • Kluck R.M.
        • Bossy-Wetzel E.
        • Green D.R.
        • Newmeyer D.D.
        The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
        Science. 1997; 275: 1132-1136
        • Yang J.
        • Liu X.
        • Bhalla K.
        • Kim C.N.
        • Ibrado A.M.
        • Cai J.
        • et al.
        Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
        Science. 1997; 275: 1129-1132
        • Lalier L.
        • Cartron P.F.
        • Juin P.
        • Nedelkina S.
        • Manon S.
        • Bechinger B.
        • et al.
        Bax activation and mitochondrial insertion during apoptosis.
        Apoptosis. 2007; 12: 887-896
        • Precht T.A.
        • Phelps R.A.
        • Linseman D.A.
        • Butts B.D.
        • Le S.S.
        • Laessig T.A.
        • et al.
        The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis.
        Cell Death Differ. 2005; 12: 255-265

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