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Alterations in Membrane Potential in Mitochondria Isolated from Brain Subregions During Focal Cerebral Ischemia and Early Reperfusion: Evaluation Using Flow Cytometry

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Abstract

Mitochondria isolated from brain tissue following middle cerebral artery occlusion or during early reperfusion were tested for their ability to generate a membrane potential under standard conditions in vitro. Membrane potential was evaluated based on rhodamine 123 fluorescence in the mitochondria as detected using flow cytometry. Compared with equivalent samples from the contralateral hemisphere, the geometric mean fluorescence was significantly lower in mitochondria prepared from the striatum and perifocal tissue in the cortex at 3 h ischemia. During reperfusion, this property was decreased in mitochondria from tissue in the striatum and cortex that had been part of severely ischemic core tissue during the arterial occlusion. These findings provide additional evidence that mitochondria develop changes during ischemia and reperfusion that are likely to limit their ability to respond to changing energy requirements and contribute to cell dysfunction and cell death. It also demonstrates the ability to gain a sensitive measure of these mitochondrial changes using flow cytometry.

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References

  1. Sims NR, Anderson MF (2002) Mitochondrial contributions to tissue damage in stroke. Neurochem Int 40:511–526. doi:10.1016/S0197-0186(01)00122-X

    Article  PubMed  CAS  Google Scholar 

  2. Hertz L (2008) Bioenergetics of cerebral ischemia: a cellular perspective. Neuropharmacology 55:289–309. doi:10.1016/j.neuropharm.2008.05.023

    Article  PubMed  CAS  Google Scholar 

  3. Folbergrova J, Memezawa H, Smith M-L et al (1992) Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats. J Cereb Blood Flow Metab 12:25–33

    PubMed  CAS  Google Scholar 

  4. Folbergrova J, Zhao Q, Katsura K et al (1995) N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia. Proc Natl Acad Sci USA 92:5057–5061. doi:10.1073/pnas.92.11.5057

    Article  PubMed  CAS  Google Scholar 

  5. Kuroda S, Katsura K-I, Hillered L et al (1996) Delayed treatment with α-phenyl-N-tert-butyl nitrone (PBN) attenuates secondary mitochondrial dysfunction after transient focal cerebral ischemia in the rat. Neurobiol Dis 3:149–157. doi:10.1006/nbdi.1996.0015

    Article  PubMed  CAS  Google Scholar 

  6. Nakai A, Kuroda S, Kristián A et al (1997) The immunosuppressant drug FK506 ameliorates secondary mitochondrial dysfunction following transient focal cerebral ischemia in the rat. Neurobiol Dis 4:288–300. doi:10.1006/nbdi.1997.0146

    Article  PubMed  CAS  Google Scholar 

  7. Anderson MF, Sims NR (1999) Mitochondrial respiratory function and cell death in focal cerebral ischemia. J Neurochem 73:1189–1199. doi:10.1046/j.1471-4159.1999.0731189.x

    Article  PubMed  CAS  Google Scholar 

  8. Keller JN, Kindy MS, Holtsberg FW et al (1998) Mitochondrial manganese superoxide dismutase prevents neuronal apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 18:687–697

    PubMed  CAS  Google Scholar 

  9. Yoshimoto T, Siesjö BK (1999) Posttreatment with the immunosuppressant cyclosporin A in transient focal ischemia. Brain Res 839:283–291. doi:10.1016/S0006-8993(99)01733-3

    Article  PubMed  CAS  Google Scholar 

  10. Matsumoto S, Friberg H, Ferrand-Drake M et al (1999) Blockade of the mitochondrial permeability transition pore diminishes infarct size in the rat after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 19:736–741. doi:10.1097/00004647-199907000-00002

    Article  PubMed  CAS  Google Scholar 

  11. Korde AS, Pettigrew LC, Craddock SD et al (2007) Protective effects of NIM811 in transient focal cerebral ischemia suggest involvement of the mitochondrial permeability transition. J Neurotrauma 24:895–908. doi:10.1089/neu.2006.0122

    Article  PubMed  Google Scholar 

  12. Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeability and cell death. Physiol Rev 87:99–163. doi:10.1152/physrev.00013.2006

    Article  PubMed  CAS  Google Scholar 

  13. Schinzel A, Takeuchi O, Huang 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 USA 102:12005–12010. doi:10.1073/pnas.0505294102

    Article  PubMed  CAS  Google Scholar 

  14. Li Y, Chopp M, Jiang N et al (1995) Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 15:389–397

    PubMed  CAS  Google Scholar 

  15. Asahi M, Hoshimaru M, Uemura Y et al (1997) Expression of interleukin-1β converting enzyme gene family and bcl-2 gene family in the rat brain following permanent occlusion of the middle cerebral artery. J Cereb Blood Flow Metab 17:11–18. doi:10.1097/00004647-199701000-00003

    Article  PubMed  CAS  Google Scholar 

  16. Fujimura M, Morita-Fujimura Y, Murakami K et al (1998) Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 18:1239–1247. doi:10.1097/00004647-199811000-00010

    Article  PubMed  CAS  Google Scholar 

  17. Namura S, Zhu JM, Fink K et al (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci 18:3659–3668

    PubMed  CAS  Google Scholar 

  18. Nicholls D, Ferguson SJ (2002) Bioenergetics 3. Academic Press, London, pp 3–8 and 57–66

  19. Duszynski J, Boguka K, Wojtczak L (1984) Homeostasis of the protonmotive force in phosphorylating mitochondria. Biochim Biophys Acta 767:540–547. doi:10.1016/0005-2728(84)90053-7

    Article  PubMed  CAS  Google Scholar 

  20. Hafner RP, Brown GC, Brand MD (1990) Analysis of the control of the respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem 188:313–319. doi:10.1111/j.1432-1033.1990.tb15405.x

    Article  PubMed  CAS  Google Scholar 

  21. Piccotti L, Marcetti C, Migliorati G et al (2002) Exogenous phospholipids specifically affect the transmembrane potential of brain mitochondria and cytochrome c release. J Biol Chem 277:12075–12081. doi:10.1074/jbc.M200029200

    Article  PubMed  CAS  Google Scholar 

  22. Carlucci A, Adornetto A, Scorziello A et al (2008) Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischemia. EMBO J 27:1073–1084. doi:10.1038/emboj.2008.33

    Article  PubMed  CAS  Google Scholar 

  23. Vega-Nunez, Alvarez AM, Menendez-Hurtado A et al (1997) Neuronal mitochondrial morphology and transmembrane potential are severely altered by hypothyroidism during rat brain development. Endocrinology 138:3771–3778. doi:10.1210/en.138.9.3771

    Article  PubMed  CAS  Google Scholar 

  24. Emaus RK, Grunwald R, Lemasters JJ (1986) Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 850:436–448. doi:10.1016/0005-2728(86)90112-X

    Article  PubMed  CAS  Google Scholar 

  25. Petit PX, O’Connor JE, Grunwald D, Brown SC (1990) Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur J Biochem 194:389–397. doi:10.1111/j.1432-1033.1990.tb15632.x

    Article  PubMed  CAS  Google Scholar 

  26. Scaduto RC, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469–477. doi:10.1016/S0006-3495(99)77214-0

    Article  PubMed  CAS  Google Scholar 

  27. Mattiasson G, Friberg H, Hansson M, Elmer E, Wieloch T (2003) Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation. J Neurochem 87:532–544. doi:10.1046/j.1471-4159.2003.02026.x

    Article  PubMed  CAS  Google Scholar 

  28. Thoren AE, Helps SC, Nilsson M et al (2005) Astrocytic function assessed from 1–14C-acetate metabolism after temporary focal cerebral ischemia in rats. J Cereb Blood Flow Metab 25:440–450. doi:10.1038/sj.jcbfm.9600035

    Article  PubMed  CAS  Google Scholar 

  29. Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17:65–134

    Article  PubMed  CAS  Google Scholar 

  30. Zea Longa EZ, Weinstein PR, Carlson S et al (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91

    Google Scholar 

  31. Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55:698–707. doi:10.1111/j.1471-4159.1990.tb04189.x

    Article  PubMed  CAS  Google Scholar 

  32. Sims NR, Anderson MF (2008) Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3:1228–1239. doi:10.1038/nprot.2008.105

    Article  PubMed  CAS  Google Scholar 

  33. Cossarizza A, Ceccarelli D, Masini A (1996) Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Exp Cell Res 222:84–94. doi:10.1006/excr.1996.0011

    Article  PubMed  CAS  Google Scholar 

  34. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  35. Phillis JW, O’Regan MH (2004) A potentially critical role of phospholipases in central nervous system ischemic, traumatic, and neurodegenerative disorders. Brain Res Brain Res Rev 44:13–47. doi:10.1016/j.brainresrev.2003.10.002

    Article  PubMed  CAS  Google Scholar 

  36. Ginsberg M (2008) Neuroprotection for ischemic stroke. Neuropharmacology 55:363–389. doi:10.1016/j.neuropharm.2007.12.007

    Article  PubMed  CAS  Google Scholar 

  37. Doyle KP, Simon RP, Stenzel-Poore MP (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55:310–318. doi:10.1016/j.neuropharm.2008.01.005

    Article  PubMed  CAS  Google Scholar 

  38. Thoren AE, Helps SC, Nilsson M et al (2006) The metabolism of 14C-glucose by neurons and astrocytes in brain subregions following focal cerebral ischemia in rats. J Neurochem 97:968–978. doi:10.1111/j.1471-4159.2006.03778.x

    Article  PubMed  CAS  Google Scholar 

  39. Bolander HG, Persson L, Hillered L, d’Argy R, Ponten U, Olsson Y (1989) Regional cerebral blood flow and histopathologic changes after middle cerebral artery occlusion in rats. Stroke 20:930–937

    PubMed  CAS  Google Scholar 

  40. Belayev L, Zhao W, Busto R et al (1997) Transient middle cerebral artery occlusion by intraluminal suture: I. Three-dimensional autoradiographic image-analysis of local cerebral glucose metabolism-blood flow interrelationships during ischemia and early recirculation. J Cereb Blood Flow Metab 17:1266–1280. doi:10.1097/00004647-199712000-00002

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), and Flinders University (Adelaide, Australia), the Rune and Ulla Amlöv Foundation and the Edit Jacobsson Foundation.

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Correspondence to Neil R. Sims.

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Lee, D.R., Helps, S.C., Macardle, P.J. et al. Alterations in Membrane Potential in Mitochondria Isolated from Brain Subregions During Focal Cerebral Ischemia and Early Reperfusion: Evaluation Using Flow Cytometry. Neurochem Res 34, 1857–1866 (2009). https://doi.org/10.1007/s11064-009-0001-1

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  • DOI: https://doi.org/10.1007/s11064-009-0001-1

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