Neurochemical Research

, Volume 38, Issue 9, pp 1880–1894 | Cite as

Nicotinamide Adenine Dinucleotide (NAD+) Repletion Attenuates Bupivacaine-Induced Neurotoxicity

  • Ting Zheng
  • Shi Yuan Xu
  • Shu Qin Zhou
  • Lu Ying Lai
  • Le Li
Original Paper


Bupivacaine is one of the most toxic local anesthetics but the mechanisms underlying its neurotoxicity are still unclear. Intracellular nicotinamide adenine dinucleotide (NAD+) depletion has been demonstrated to play an essential role in neuronal injury. In the present study, we investigated whether intracellular NAD+ depletion contributes to bupivacaine-induced neuronal injury and whether NAD+ repletion attenuates the injury in SH-SY5Y cells. First, we evaluated the intracellular NAD+ content after bupivacaine exposure. We also examined the cellular NAD+ level after pretreatment with exogenous NAD+. We next determined cell viability and the apoptosis rate after bupivacaine treatment in the presence or absence of NAD+ incubation. Finally, cell injuries such as nuclear injury, reactive oxygen species (ROS) production, and mitochondrial depolarization were detected after bupivacaine treatment with or without NAD+ pretreatment. Bupivacaine caused intracellular NAD+ depletion in a time- and concentration-dependent manner. Cellular NAD+ replenishment prevented cell death and apoptosis induced by bupivacaine. Importantly, exogenous NAD+ attenuated bupivacaine-induced nuclear injury, ROS production, and mitochondrial depolarization. Our results suggest that NAD+ depletion is necessary for bupivacaine-induced neuronal necrosis and apoptosis, and that NAD+ repletion attenuates neurotoxicity resulting from bupivacaine-treatment.


NAD+  Neurotoxicity  Bupivacaine  Energy metabolism 



We express our gratitude to Miss Hong Fen Shen and Mr. Yong Bing Ye for practical assistance. We thank Mr. Chun Hong Jia for his constructive comments regarding the manuscript. This study was supported by the grants from the National Natural Science Foundation of China (no. 81271390) and Natural Science Foundation of Guangdong Province, China (no. s2011010004056). All of the authors have disclosed no financial relationship with a biotechnology manufacturer, a pharmaceutical company, or other commercial entities with an interest in subject matter or materials discussed in the manuscripts.


  1. 1.
    Perez-Castro R, Patel S, Garavito-Aguilar ZV, Rosenberg A, Recio-Pinto E, Zhang J, Blanck TJJ, Xu F (2009) Cytotoxicity of local anesthetics in human neuronal cells. Anesth Analg 108(3):997–1007. doi: 10.1213/ane.0b013e31819385e1 PubMedCrossRefGoogle Scholar
  2. 2.
    Ma R, Wang X, Lu C, Li C, Cheng Y, Ding G, Liu L, Ding Z (2010) Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine–serine protein kinase B-dependent mechanism. Neuroscience 167(2):329–342. doi: 10.1016/j.neuroscience.2009.12.049 PubMedCrossRefGoogle Scholar
  3. 3.
    Lu J, Xu SY, Zhang QG, Xu R, Lei HY (2011) Bupivacaine induces apoptosis via mitochondria and p38 MAPK dependent pathways. Eur J Pharmacol 657(1–3):51–58. doi: 10.1016/j.ejphar.2011.01.055 PubMedCrossRefGoogle Scholar
  4. 4.
    Lirk P, Haller I, Peter H, Lang L, Tomaselli B, Klimaschewski L, Gerner P (2008) In vitro, inhibition of mitogen-activated protein kinase pathways protects against bupivacaine- and ropivacaine-induced neurotoxicity. Anesth Analg 106(5):1456–1464. doi: 10.1213/ane.0b013e318168514b PubMedCrossRefGoogle Scholar
  5. 5.
    Park CJ, Park SA, Yoon TG, Lee SJ, Yum KW, Kim HJ (2005) Bupivacaine induces apoptosis via ROS in the schwann cell line. J Dent Res 84(9):852–857. doi: 10.1177/154405910508400914 PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson ME, Saenz JA, DaSilva AD, Uhl CB, Gores GJ (2002) Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model. Anesthesiology 97(6):1466–1476PubMedCrossRefGoogle Scholar
  7. 7.
    Liu D, Gharavi R, Pitta M, Gleichmann M, Mattson MP (2009) Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons. Neuromolecular Med 11(1):28–42. doi: 10.1007/s12017-009-8058-1 PubMedCrossRefGoogle Scholar
  8. 8.
    Mattson MP, Liu D (2002) Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders. Neuromolecular Med 2(2):215–231. doi: 10.1385/nmm:2:2:215 PubMedCrossRefGoogle Scholar
  9. 9.
    Sasaki Y, Vohra BPS, Lund FE, Milbrandt J (2009) Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide. J Neurosci 29(17):5525–5535. doi: 10.1523/jneurosci.5469-08.2009 PubMedCrossRefGoogle Scholar
  10. 10.
    Wang J (2005) A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol 170(3):349–355. doi: 10.1083/jcb.200504028 PubMedCrossRefGoogle Scholar
  11. 11.
    Alano CC (2004) Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition. J Biol Chem 279(18):18895–18902. doi: 10.1074/jbc.M313329200 PubMedCrossRefGoogle Scholar
  12. 12.
    Araki T (2004) Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 305(5686):1010–1013. doi: 10.1126/science.1098014 PubMedCrossRefGoogle Scholar
  13. 13.
    Ying W (2008) NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal 10(2):179–206. doi: 10.1089/ars.2007.1672 PubMedCrossRefGoogle Scholar
  14. 14.
    Kim SH, Lu HF, Alano CC (2011) Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture. PLoS ONE 6(3):e14731. doi: 10.1371/journal.pone.0014731 PubMedCrossRefGoogle Scholar
  15. 15.
    Alano CC, Garnier P, Ying W, Higashi Y, Kauppinen TM, Swanson RA (2010) NAD+ depletion is necessary and sufficient for Poly(ADP-Ribose) polymerase-1-mediated neuronal death. J Neurosci 30(8):2967–2978. doi: 10.1523/jneurosci.5552-09.2010 PubMedCrossRefGoogle Scholar
  16. 16.
    Kaundal RK, Shah KK, Sharma SS (2006) Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation. Life Sci 79(24):2293–2302. doi: 10.1016/j.lfs.2006.07.034 PubMedCrossRefGoogle Scholar
  17. 17.
    Liu D, Pitta M, Mattson MP (2008) Preventing NAD+ depletion protects neurons against excitotoxicity. Ann N Y Acad Sci 1147(1):275–282. doi: 10.1196/annals.1427.028 PubMedCrossRefGoogle Scholar
  18. 18.
    Yan T, Feng Y, Zheng J, Ge X, Zhang Y, Wu D, Zhao J, Zhai Q (2010) Nmnat2 delays axon degeneration in superior cervical ganglia dependent on its NAD synthesis activity. Neurochem Int 56(1):101–106. doi: 10.1016/j.neuint.2009.09.007 PubMedCrossRefGoogle Scholar
  19. 19.
    Ying W, Wei G, Wang D, Wang Q, Tang X, Shi J, Zhang P, Lu H (2007) Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia. Front Biosci 12:2728–2734PubMedCrossRefGoogle Scholar
  20. 20.
    Pillai VB, Sundaresan NR, Kim G, Gupta M, Rajamohan SB, Pillai JB, Samant S, Ravindra PV, Isbatan A, Gupta MP (2010) Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway. J Biol Chem 285(5):3133–3144. doi: 10.1074/jbc.M109.077271 PubMedCrossRefGoogle Scholar
  21. 21.
    Wang J, He Z (2009) NAD and axon degeneration: from the Wlds gene to neurochemistry. Cell Adh Migr 3(1):77–87PubMedCrossRefGoogle Scholar
  22. 22.
    Ying W, Garnier P, Swanson RA (2003) NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes. Biochem Biophys Res Commun 308(4):809–813. doi: 10.1016/s0006-291x(03)01483-9 PubMedCrossRefGoogle Scholar
  23. 23.
    Zong WX (2004) Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev 18(11):1272–1282. doi: 10.1101/gad.1199904 PubMedCrossRefGoogle Scholar
  24. 24.
    Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, Dawson VL (2006) Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci 103(48):18314–18319. doi: 10.1073/pnas.0606528103 PubMedCrossRefGoogle Scholar
  25. 25.
    D’Amours D, Desnoyers S, D’Silva I, Poirier GG (1999) Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J 342(Pt 2):249–268PubMedCrossRefGoogle Scholar
  26. 26.
    Virag L, Szabo C (2002) The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev 54(3):375–429PubMedCrossRefGoogle Scholar
  27. 27.
    Beilin Y, Halpern S (2010) Ropivacaine versus bupivacaine for epidural labor analgesia. Anesth Analg 111(2):482–487. doi: 10.1213/ANE.0b013e3181e3a08e PubMedCrossRefGoogle Scholar
  28. 28.
    Chan VW, Peng P, Chinyanga H, Lazarou S, Weinbren J, Kaszas Z (2000) Determining minimum effective anesthetic concentration of hyperbaric bupivacaine for spinal anesthesia. Anesth Analg 90(5):1135–1140PubMedCrossRefGoogle Scholar
  29. 29.
    Wang J, Zhai Q, Chen Y, Lin E, Gu W, McBurney MW, He Z (2005) A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol 170(3):349–355. doi: 10.1083/jcb.200504028 PubMedCrossRefGoogle Scholar
  30. 30.
    Xie HR, Hu LS, Li GY (2010) SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson’s disease. Chin Med J (Engl) 123(8):1086–1092Google Scholar
  31. 31.
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184(1):39–51PubMedCrossRefGoogle Scholar
  32. 32.
    Span LF, Pennings AH, Vierwinden G, Boezeman JB, Raymakers RA, de Witte T (2002) The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique. Cytometry 47(1):24–31PubMedCrossRefGoogle Scholar
  33. 33.
    Wang S, Xing Z, Vosler PS, Yin H, Li W, Zhang F, Signore AP, Stetler RA, Gao Y, Chen J (2008) Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced dna repair. Stroke 39(9):2587–2595. doi: 10.1161/strokeaha.107.509158 PubMedCrossRefGoogle Scholar
  34. 34.
    Unami A, Shinohara Y, Ichikawa T, Baba Y (2003) Biochemical and microarray analyses of bupivacaine-induced apoptosis. J Toxicol Sci 28(2):77–94PubMedCrossRefGoogle Scholar
  35. 35.
    Lu J, Xu SY, Zhang QG, Lei HY (2011) Bupivacaine induces reactive oxygen species production via activation of the amp-activated protein kinase-dependent pathway. Pharmacology 87(3–4):121–129. doi: 10.1159/000323402 PubMedCrossRefGoogle Scholar
  36. 36.
    Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594:57–72. doi: 10.1007/978-1-60761-411-1_4 PubMedCrossRefGoogle Scholar
  37. 37.
    Salido M, Gonzalez JL, Vilches J (2007) Loss of mitochondrial membrane potential is inhibited by bombesin in etoposide-induced apoptosis in PC-3 prostate carcinoma cells. Mol Cancer Ther 6(4):1292–1299. doi: 10.1158/1535-7163.mct-06-0681 PubMedCrossRefGoogle Scholar
  38. 38.
    Fakler M, Loeder S, Vogler M, Schneider K, Jeremias I, Debatin KM, Fulda S (2009) Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance. Blood 113(8):1710–1722. doi: 10.1182/blood-2007-09-114314 PubMedCrossRefGoogle Scholar
  39. 39.
    Ciccarone V, Spengler BA, Meyers MB, Biedler JL, Ross RA (1989) Phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages. Cancer Res 49(1):219–225PubMedGoogle Scholar
  40. 40.
    Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, Poirier GG, Dawson TM, Dawson VL (2002) Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297(5579):259–263. doi: 10.1126/science.1072221 PubMedCrossRefGoogle Scholar
  41. 41.
    Ying W, Sevigny MB, Chen Y, Swanson RA (2001) Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death. Proc Natl Acad Sci 98(21):12227–12232. doi: 10.1073/pnas.211202598 PubMedCrossRefGoogle Scholar
  42. 42.
    Olek RA, Ziolkowski W, Kaczor JJ, Greci L, Popinigis J, Antosiewicz J (2004) Antioxidant activity of NADH and its analogue: an in vitro study. J Biochem Mol Biol 37(4):416–421PubMedCrossRefGoogle Scholar
  43. 43.
    Quintana-Cabrera R, Bolanos JP (2013) Glutathione and γ-glutamylcysteine in the antioxidant and survival functions of mitochondria. Biochem Soc Trans 41(1):106–110. doi: 10.1042/BST20120252 PubMedCrossRefGoogle Scholar
  44. 44.
    Shi F, Li Y, Li Y, Wang X (2009) Molecular properties, functions, and potential applications of NAD kinases. Acta Biochim Biophys Sin 41(5):352–361. doi: 10.1093/abbs/gmp029 PubMedCrossRefGoogle Scholar
  45. 45.
    Zhang H, Forman HJ (2012) Glutathione synthesis and its role in redox signaling. Semin Cell Dev Biol 23(7):722–728. doi: 10.1016/j.semcdb.2012.03.017 PubMedCrossRefGoogle Scholar
  46. 46.
    Lu SC (2009) Regulation of glutathione synthesis. Mol Aspects Med 30(1–2):42–59. doi: 10.1016/j.mam.2008.05.005 PubMedCrossRefGoogle Scholar
  47. 47.
    Hipkiss AR (2009) NAD+ availability and proteotoxicity. Neuromolecular Med 11(2):97–100. doi: 10.1007/s12017-009-8069-y PubMedCrossRefGoogle Scholar
  48. 48.
    Desai KM, Wu L (2008) Free radical generation by methylglyoxal in tissues. Drug Metabol Drug Interact 23(1–2):151–173PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ting Zheng
    • 1
  • Shi Yuan Xu
    • 1
  • Shu Qin Zhou
    • 1
  • Lu Ying Lai
    • 1
  • Le Li
    • 1
  1. 1.Department of Anesthesiology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina

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