All Title Author
Keywords Abstract

PLOS ONE  2012 

Reperfusion Promotes Mitochondrial Dysfunction following Focal Cerebral Ischemia in Rats

DOI: 10.1371/journal.pone.0046498

Full-Text   Cite this paper   Add to My Lib


Background and Purpose Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia. Methods Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis. Results Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca2+ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period. Conclusions Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca2+-induced mitochondrial swelling. The mechanism of this swelling may be mediated by the upregulation of the Cyclophilin D protein, the destruction of the mitochondrial membrane potential and the generation of excessive reactive oxidative species.


[1]  Doyle KP, Simon RP, Stenzel-Poore MP (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55: 310–318.
[2]  Lust WD, Taylor C, Pundik S, Selman WR, Ratcheson RA (2002) Ischemic cell death: dynamics of delayed secondary energy failure during reperfusion following focal ischemia. Metab Brain Dis 17: 113–121.
[3]  White BC, Sullivan JM, DeGracia DJ, O'Neil BJ, Neumar RW, et al. (2000) Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J Neurol Sci 179: 1–33.
[4]  Schaller B, Graf R (2004) Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab 24: 351–71.
[5]  Anderson MF, Sims NR (1999) Mitochondrial respiratory function and cell death in focal cerebral ischemia. J Neurochem 73: 1189–1199.
[6]  Sims NR, Anderson MF (2002) Mitochondrial contributions to tissue damage in stroke. Neurochem Int 40: 511–526.
[7]  Murakami K, Kondo T, Kawase M, Li Y, Sato S, et al. (1998) Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci 18: 205–213.
[8]  Garcia JH, Lossinsky AS, Kauffman FC, Conger KA (1978) Neuronal ischemic injury: light microscopy, ultrastructure and biochemistry. Acta Neuropathol 43: 85–95.
[9]  Garcia JH (1975) The neuropathology of stroke. Hum Pathol 6: 583–98.
[10]  Kalimo H, Garcia JH, Kamijyo Y, Tanaka J, Trump BF (1977) The ultrastructure of “brain death”. II. Electron microscopy of feline cortex after complete ischemia. Virchows Arch B Cell Pathol 25: 207–20.
[11]  Murphy AN, Fiskum G, Beal MF (1999) Mitochondria in neurodegeneration: bioenergetic function in cell life and death. J Cereb Blood Flow Metab 19: 231–45.
[12]  Tsujimoto Y, Nakagawa T, Shimizu S (2006) Mitochondrial membrane permeability transition and cell death. Biochim Biophys Acta 1757: 1297–1300.
[13]  Kristián T, Siesj? BK (1998) Calcium in ischemic cell death. Stroke 29: 705–18.
[14]  Hansson MJ, Persson T, Friberg H, Keep MF, Rees A, et al. (2003) Powerful cyclosporin inhibition of calcium-induced permeability transition in brain mitochondria. Brain Res 960: 99–111.
[15]  Kobayashi T, Kuroda S, Tada M, Houkin K, Iwasaki Y, et al. (2003) Calcium-induced mitochondrial swelling and cytochrome c release in the brain: its biochemical characteristics and implication in ischemic neuronal injury. Brain Res 960: 62–70.
[16]  Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233–49.
[17]  Halestrap AP, McStay GP, Clarke SJ (2002) The permeability transition pore complex: another view. Biochimie 84: 153–66.
[18]  Naga KK, Sullivan PG, Geddes JW (2007) High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J Neurosci 27: 7469–75.
[19]  Schinzel AC, Takeuchi O, Huang Z, Fisher JK, Zhou Z, et al. (2005) Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl Acad Sci U S A 102: 12005–10.
[20]  Zaidan E, Sims NR (1997) Reduced activity of the pyruvate dehydrogenase complex but not cytochrome c oxidase is associated with neuronal loss in the striatum following short-term forebrain ischemia. Brain Res 772: 23–8.
[21]  Canevari L, Kuroda S, Bates TE, Clark JB, Siesj? BK (1997) Activity of mitochondrial respiratory chain enzymes after transient focal ischemia in the rat. J Cereb Blood Flow Metab 17: 1166–9.
[22]  Zoratti M, Szabò I (1995) The mitochondrial permeability transition. Biochim Biophys Acta 1241: 139–76.
[23]  Iijima T (2006) Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 55: 234–43.
[24]  Hou ST, MacManus JP (2002) Molecular mechanisms of cerebral ischemia-induced neuronal death. Int Rev Cytol 221: 93–148.
[25]  Niizuma K, Endo H, Chan PH (2009) Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem 1: 133–8.
[26]  Kim GW, Kondo T, Noshita N, Chan PH (2002) Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke 33: 809–815.
[27]  Piantadosi CA, Zhang J (1996) Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke 27: 327–31.
[28]  Feng ZC, Sick TJ, Rosenthal M (1998) Oxygen sensitivity of mitochondrial redox status and evoked potential recovery early during reperfusion in post-ischemic rat brain. Resuscitation 37: 33–41.
[29]  Fiskum G, Rosenthal RE, Vereczki V, Martin E, Hoffman GE, et al. (2004) Protection against ischemic brain injury by inhibition of mitochondrial oxidative stress. J Bioenerg Biomembr 36: 347–52.
[30]  Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20: 84–91.
[31]  Shimamura N, Matchett G, Tsubokawa T, Ohkuma H, Zhang J (2006) Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model. J Neurosci Methods 156: 161–165.
[32]  Sims NR, Anderson MF (2008) Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3: 1228–1239.
[33]  Hansson MJ, Mattiasson G, M?nsson R, Karlsson J, Keep MF, et al. (2004) The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria. J Bioenerg Biomembr 36: 407–413.
[34]  Adembri C, Venturi L, Tani A, Chiarugi A, Gramigni E, et al. (2006) Neuroprotective effects of propofol in models of cerebral ischemia: inhibition of mitochondrial swelling as a possible mechanism. Anesthesiology 104: 80–89.
[35]  Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55: 698–707.
[36]  Peinado MA, Quesada A, Pedrosa JA, Martinez M, Esteban FJ (1997) Light microscopic quantification of morphological changes during aging in neurons and glia of the rat parietal cortex. Anat Rec 247: 420–5.
[37]  Popp A, Jaenisch N, Witte OW, Frahm C (2009) Identification of ischemic regions in a rat model of stroke. PLoS One 4: e4764.
[38]  McGee-Russell SM, Brown AW, Brierley JB (1970) A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain. Brain Res 20: 193–200.
[39]  Brown AW, Brierley JB (1972) Anoxic-ischaemic cell change in rat brain light microscopic and fine-structural observations. J Neurol Sci 16: 59–84.
[40]  Solenski NJ, diPierro CG, Trimmer PA, Kwan AL, Helm GA (2002) Ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia. Stroke 33: 816–824.
[41]  Kristal BS, Dubinsky JM (1997) Mitochondrial permeability transition in the central nervous system: induction by calcium cycling-dependent and -independent pathways. J Neurochem 69: 524–38.
[42]  Schild L, Reiser G (2005) Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+. FEBS J 272: 3593–601.
[43]  Wang X, Carlsson Y, Basso E, Zhu C, Rousset CI, et al. (2009) Developmental shift of cyclophilin D contribution to hypoxic-ischemic brain injury. J Neurosci 29: 2588–96.
[44]  Sims NR, Zaidan E (1995) Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int J Biochem Cell Biol 27: 531–50.
[45]  Iijima T, Mishima T, Tohyama M, Akagawa K, Iwao Y (2003) Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron. Neurochem Int 43: 263–9.
[46]  Iijima T, Mishima T, Akagawa K, Iwao Y (2003) Mitochondrial hyperpolarization after transient oxygen-glucose deprivation and subsequent apoptosis in cultured rat hippocampal neurons. Brain Res 993: 140–5.
[47]  Nicholls DG, Budd SL (2000) Mitochondria and neuronal survival. Physiol Rev 80: 315–60.
[48]  Myers KM, Fiskum G, Liu Y, Simmens SJ, Bredesen DE, et al. (1995) Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability. J Neurochem 65: 2432–40.
[49]  Liu Y, Rosenthal RE, Haywood Y, Miljkovic-Lolic M (1998) Normoxic ventilation after cardiac arrest reduces oxidation of brain lipids and improves neurological outcome. Stroke 29: 1679–86.
[50]  Solenski NJ, Kwan AL, Yanamoto H, Bennett JP, Kassell NF, et al. (1997) Differential hydroxylation of salicylate in core and penumbra regions during focal reversible cerebral ischemia. Stroke 28: 2545–51.
[51]  Kowaltowski AJ, Vercesi AE, Fiskum G (2000) Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides. Cell Death Differ 7: 903–10.
[52]  Borutaite V, Morkuniene R, Brown GC (1999) Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca2+-induced inhibition of substrate oxidation. Biochim Biophys Acta 1999 1453: 41–8.


comments powered by Disqus