Detection of Mitochondrial Reactive Oxygen Species in Living Rat Trigeminal Caudal Neurons

  • Lee, Hae In (Dept. of Oral Physiology, College of Dentistry, Institute of Wonkwang Biomaterial and Implant, Wonkwang University) ;
  • Chun, Sang Woo (Dept. of Oral Physiology, College of Dentistry, Institute of Wonkwang Biomaterial and Implant, Wonkwang University)
  • Received : 2015.05.29
  • Accepted : 2015.06.15
  • Published : 2015.06.30


Growing evidence suggests that mitochondrial reactive oxygen species (ROS) are involved in various pain states. This study was performed to investigate whether ROS-induced changes in neuronal excitability in trigeminal subnucleus caudalis are related to ROS generation in mitochondria. Confocal scanning laser microscopy was used to measure ROS-induced fluorescence intensity in live rat trigeminal caudalis slices. The ROS level increased during the perfusion of malate, a mitochondrial substrate, after loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), an indicator of the intracellular ROS; the ROS level recovered to the control condition after washout. When pre-treated with phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), malate-induced increase of ROS level was suppressed. To identify the direct relation between elevated ROS levels and mitochondria, we applied the malate after double-loading of $H_2DCF-DA$ and chloromethyl-X-rosamine (CMXRos; MitoTracker Red), which is a mitochondria-specific fluorescent probe. As a result, increase of both intracellular ROS and mitochondrial ROS were observed simultaneously. This study demonstrated that elevated ROS in trigeminal subnucleus caudalis neuron can be induced through mitochondrial-ROS pathway, primarily by the leakage of ROS from the mitochondrial electron transport chain.


Supported by : 원광대학교


  1. Dubner R, Bennett GJ. Spinal and trigeminal mechanisms of nociception. Annu Rev Neurosci. 1983;6:381-418.
  2. Sessle BJ. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit Rev Oral Biol Med. 2000;11:57-91.
  3. Levy D, Zochodne DW. Local nitric oxide synthase activity in a model of neuropathic pain. Eur J Neurosci. 1998;10:1846-1855.
  4. Khalil Z, Khodr B. A role for free radicals and nitric oxide in delayed recovery in aged rats with chronic constriction nerve injury. Free Rad Biol Med. 2001;31:430-439.
  5. Liu D, Liu J, Sun D, Wen J. The time course of hydroxyl radical formation following spinal cord injury: the possible role of the iron-catalyzed Haber-Weiss reaction. J Neurotrauma 2004;21:805-816.
  6. Wang ZQ, Porreca F, Cuzzocrea S, Galen K, Lightfoot R, Masini E. A newly identified role for superoxide in inflammatory pain. J Pharmacol Exp Ther. 2004;309:869-878.
  7. Djordjevic VB. Free radicals in cell biology. Int Rev Cytol. 2004;237:57-89.
  8. McGeer EG, McGeer PL. Brain inflammation in Alzheimer disease and the therapeutic implications. Curr Pharm Res. 1999;5:821-836.
  9. Wells PG, Kim PM, Laposa RR, Nicol CJ, Parman T, Winn LM. Oxidative damage in chemical teratogenesis. Mutat Res. 1997;396:65-78.
  10. Parman T, Wiley MJ, Wells PG. Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat Med. 1999;5:582-585.
  11. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS induced ROS release: an update and review. Biochim Biophys Acta. 2006;1757:509-517.
  12. Kim HY, Chung JM, Chung K. Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: the effect of mitochondrial electron transport complex inhibitors. Neurosci Lett. 2008;447:87-91. doi:10.1016/j.neulet.2008.09.041.
  13. Stowe DF, Camara AK. Mitochondrial reactive oxygen species production in excitable cells: modulators of mitochondrial and cell function. Antioxid Redox Signal. 2009;11:1373-1414. doi:10.1089/ARS.2008.2331.
  14. Park ES, Gao X, Chung JM, Chung K. Levels of mitochondrial reactive oxygen species increase in rat neuropathic spinal dorsal horn neurons. Neurosci Lett. 2006;391:108-111.
  15. Schwartz ES, Kim HY, Wang J, Lee I, Klann E, Chung JM, Chung K. Persistent pain is dependent on spinal mitochondrial antioxidant levels. J Neurosci. 2008;29:159-168. doi:10.1523/JNEUROSCI.3792-08.2009.
  16. Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, Chung JM. Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain 2004;111:116-124.
  17. Khattab MM. TEMPOL, a membrane-permeable radical scavenger, attenuates peroxynitrite-and superoxide anion enhanced carrageenan-induced paw edema and hyperalgesia: a key role for superoxide anion. Eur J Pharmacol. 2006;548:167-173.
  18. Lee I, Kim HK, Kim JH, Chung K, Chung JM. The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons. Pain 2007;133:9-17.
  19. Gonzalez C, Sanz-Alfayate G, Agapito MT, Gomez-Nino A, Rocher A, Obeso A. Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia. Respir Physiol Neurobiol. 2002;132:17-41.
  20. Baran CP, Zeigler MM, Tridandapani S, Marsh CB. The role of ROS and RNA in regulating life and death of blood monocytes. Curr Pharm. 2004;10:855-866.
  21. Bubici C, Papa S, Pham CG, Zazzeroni F, Franzoso G. The NF-kappaB-mediated control of ROS and JNK signaling. Histol Histopathol. 2006;21:69-80.
  22. Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, Hoppel CL. Mitochondrial dysfunction in cardiac disease: ischemiareperfusion, aging, and heart failure. J Mol Cell Cardiol. 2001;33:1065-1089.
  23. Zoccarato F, Cavallini L, Bortolami S, Alexandre A. Succinate modulation of $H_2O_2$ release at NADH: ubiquinone oxidore ductase (Complex I) in brain mitochondria. Biochem J. 2007;406:125-129.
  24. Verkhovskaya ML, Belevich N, Euro L, Wikstrom M, Verkhovsky MI. Real-time electron transfer in respiratory complex I. Proc Natl Acad Sci USA. 2008;105:3763-3767. doi:10.1073/pnas.0711249105.
  25. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem. 2003;278: 36027-36031.
  26. Hoffman DL, Brookes PS. Oxygen sensitivity of mitochondrial reactive oxygen species generation depends on metabolic conditions. J Biol Chem. 2009;284:16236-16245. doi:10.1074/jbc.M809512200.
  27. LeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992;5:227-231.
  28. Wang J, Li G, Wang Z, Zhang X, Yao L, Wang F, Liu S, Yin J, Ling EA, Wang L, Hao A. High glucose-induced expression of inflammatory cytokines and reactive oxygen species in cultured astrocytes. Neuroscience 2012; 202:58-68. doi:10.1016/j.neuroscience.2011.11.062.
  29. Lee HI, Park AR, Chun SW. Effects of NaOCl on Neuronal Excitability and Intracellular Calcium Concentration in Rat Spinal Substantia Gelatinosa Neurons. Int J Oral Biol. 2013;38:5-12. doi:
  30. Gwak YS, Hassler SE, Hulsebosch CE. Reactive oxygen species contribute to neuropathic pain and locomotor dysfunction via activation of CamKII in remote segments following spinal cord contusion injury in rats. Pain 2013;154:1699-1708. doi:10.1016/j.pain.2013.05.018.
  31. Avshalumov MV, Chen BT, Marshall SP, Pena DM, Rice ME. Glutamate-dependent inhibition of dopamine release in striatum is mediated by a new diffusible messenger, $H_2O_2$. J Neurosci. 2003;23:2744-2750.
  32. Bao L, Avshalumov MV, Rice ME. Partial mitochondrial inhibition causes striatal dopamine release suppression and medium spiny neuron depolarization via $H_2O_2$ elevation, not ATP depletion. J Neurosci. 2005;25:10029-10040.
  33. Avshalumov MV, Chen BT, Koos T, Rice ME. Endogenous hydrogen peroxide regulates the excitability of midbrain dopamine neurons via ATP-sensitive potassium channels. J Neurosci. 2005;25:4222-4231.
  34. Shanker G, Aschner JL, Syversen T, Aschner M. Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury. Mol Brain Res. 2004;128:48-57.
  35. Hempel SL, Buettner GR, O'Malley YQ, Wessels DA, Flaherty DM. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med. 1999;27:146-159.
  36. Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol. 1994;55:253-258.
  37. Lee SB, Bae IH, Bae YS, Um HD. Link between mitochondria and NADPH oxidase 1 isozyme for the sustained production of reactive oxygen species and cell death. J Biol Chem. 2006;281:36228-36235.