Elevated Neuroglobin Lessens Neuroinflammation and Alleviates Neurobehavioral Deficits Induced by Acute Inhalation of Combustion Smoke in the Mouse

  • Murat F. Gorgun
  • Ming Zhuo
  • Kelly T. Dineley
  • Ella W. EnglanderEmail author
Original Paper


Acute inhalation of combustion smoke produces long-term neurologic deficits in survivors. To study the mechanisms that contribute to the development of neurologic deficits and identify targets for prevention, we developed a mouse model of acute inhalation of combustion smoke, which supports longitudinal investigation of mechanisms that underlie the smoke induced inimical sequelae in the brain. Using a transgenic mouse engineered to overexpress neuroglobin, a neuroprotective oxygen-binding globin protein, we previously demonstrated that elevated neuroglobin preserves mitochondrial respiration and attenuates formation of oxidative DNA damage in the mouse brain after smoke exposure. In the current study, we show that elevated neuronal neuroglobin attenuates the persistent inflammatory changes induced by smoke exposure in the mouse brain and mitigates concordant smoke-induced long-term neurobehavioral deficits. Specifically, we found that increases in hippocampal density of GFAP and Iba-1 positive cells that are detected post-smoke in wild-type mice are absent in the neuroglobin overexpressing transgenic (Ngb-tg) mice. Similarly, the smoke induced hippocampal myelin depletion is not observed in the Ngb-tg mice. Importantly, elevated neuroglobin alleviates behavioral and memory deficits that develop after acute smoke inhalation in the wild-type mice. Taken together, our findings suggest that the protective effects exerted by neuroglobin in the brains of smoke exposed mice afford protection from long-term neurologic sequelae of acute inhalation of combustion smoke. Our transgenic mouse provides a tool for assessing the potential of elevated neuroglobin as possible strategy for management of smoke inhalation injury.


Neuroglobin Neuroprotection Neuroinflammation Combustion smoke inhalation brain injury Neurogenesis Novel object recognition 



This work was supported by grants from Shriners Hospitals for Children (86700) and the National Institutes of Health (ES014613) to EWE.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Burmester T, Weich B, Reinhardt S, Hankeln T (2000) A vertebrate globin expressed in the brain. Nature 407:520–523CrossRefGoogle Scholar
  2. 2.
    Droge J, Pande A, Englander EW, Makalowski W (2012) Comparative genomics of neuroglobin reveals its early origins. PLoS ONE 7:e47972CrossRefGoogle Scholar
  3. 3.
    Hundahl CA, Allen GC, Hannibal J, Kjaer K, Rehfeld JF, Dewilde S, Nyengaard JR, Kelsen J, Hay-Schmidt A (2010) Anatomical characterization of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain. Brain Res 1331:58–73CrossRefGoogle Scholar
  4. 4.
    Hundahl CA, Allen GC, Nyengaard JR, Dewilde S, Carter BD, Kelsen J, Hay-Schmidt A (2008) Neuroglobin in the rat brain: localization. Neuroendocrinology 88:173–182CrossRefGoogle Scholar
  5. 5.
    Van Acker ZP, Luyckx E, Dewilde S (2019) Neuroglobin expression in the brain: a story of tissue homeostasis preservation. Mol Neurobiol 56:2101–2122CrossRefGoogle Scholar
  6. 6.
    Burmester T, Hankeln T (2009) What is the function of neuroglobin? J Exp Biol 212:1423–1428CrossRefGoogle Scholar
  7. 7.
    Baez E, Echeverria V, Cabezas R, Avila-Rodriguez M, Garcia-Segura LM, Barreto GE (2016) Protection by neuroglobin expression in brain pathologies. Front Neurol 7:146CrossRefGoogle Scholar
  8. 8.
    Guidolin D, Tortorella C, Marcoli M, Maura G, Agnati LF (2016) Neuroglobin, a factor playing for nerve cell survival. Int J Mol Sci 17:1817CrossRefGoogle Scholar
  9. 9.
    Azarov I, Wang L, Rose JJ, Xu Q, Huang XN, Belanger A, Wang Y, Guo L, Liu C, Ucer KB, McTiernan CF, O'Donnell CP, Shiva S, Tejero J, Kim-Shapiro DB, Gladwin MT (2016) Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. Sci Transl Med 8:368ra173CrossRefGoogle Scholar
  10. 10.
    Brittain T, Skommer J (2012) Does a redox cycle provide a mechanism for setting the capacity of neuroglobin to protect cells from apoptosis? IUBMB Life 64:419–422CrossRefGoogle Scholar
  11. 11.
    Hundahl CA, Fahrenkrug J, Hay-Schmidt A, Georg B, Faltoft B, Hannibal J (2012) Circadian behaviour in neuroglobin deficient mice. PLoS ONE 7:e34462CrossRefGoogle Scholar
  12. 12.
    Hundahl CA, Kelsen J, Dewilde S, Hay-Schmidt A (2008) Neuroglobin in the rat brain (II): co-localisation with neurotransmitters. Neuroendocrinology 88:183–198CrossRefGoogle Scholar
  13. 13.
    Khan AA, Wang Y, Sun Y, Mao XO, Xie L, Miles E, Graboski J, Chen S, Ellerby LM, Jin K, Greenberg DA (2006) Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia. Proc Natl Acad Sci USA 103:17944–17948CrossRefGoogle Scholar
  14. 14.
    Kiger L, Tilleman L, Geuens E, Hoogewijs D, Lechauve C, Moens L, Dewilde S, Marden MC (2011) Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom. PLoS ONE 6:e20478CrossRefGoogle Scholar
  15. 15.
    Li W, Wu Y, Ren C, Lu Y, Gao Y, Zheng X, Zhang C (2011) The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger. Proteins 79:115–125CrossRefGoogle Scholar
  16. 16.
    Singh S, Zhuo M, Gorgun FM, Englander EW (2013) Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons. Nitric Oxide 32:21–28CrossRefGoogle Scholar
  17. 17.
    Sun Y, Jin K, Peel A, Mao XO, Xie L, Greenberg DA (2003) Neuroglobin protects the brain from experimental stroke in vivo. Proc Natl Acad Sci USA 100:3497–3500CrossRefGoogle Scholar
  18. 18.
    Lee HM, Greeley GH Jr, Englander EW (2011) Transgenic overexpression of neuroglobin attenuates formation of smoke-inhalation-induced oxidative DNA damage, in vivo, in the mouse brain. Free Radic Biol Med 51:2281–2287CrossRefGoogle Scholar
  19. 19.
    Lee HM, Greeley GH, Herndon DN, Sinha M, Luxon BA, Englander EW (2005) A rat model of smoke inhalation injury: influence of combustion smoke on gene expression in the brain. Toxicol Appl Pharmacol 208:255–265CrossRefGoogle Scholar
  20. 20.
    Lee HM, Hallberg LM, Greeley GH Jr, Englander EW (2010) Differential inhibition of mitochondrial respiratory complexes by inhalation of combustion smoke and carbon monoxide, in vivo, in the rat brain. Inhal Toxicol 22:770–777CrossRefGoogle Scholar
  21. 21.
    Lee HM, Reed J, Greeley GH Jr, Englander EW (2009) Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke. Toxicol Appl Pharmacol 235:208–215CrossRefGoogle Scholar
  22. 22.
    Hartzell GE (1996) Overview of combustion toxicology. Toxicology 115:7–23CrossRefGoogle Scholar
  23. 23.
    Rossi J 3rd, Ritchie GD, Macys DA, Still KR (1996) An overview of the development, validation, and application of neurobehavioral and neuromolecular toxicity assessment batteries: potential applications to combustion toxicology. Toxicology 115:107–117CrossRefGoogle Scholar
  24. 24.
    Stefanidou M, Athanaselis S, Spiliopoulou C (2008) Health impacts of fire smoke inhalation. Inhal Toxicol 20:761–766CrossRefGoogle Scholar
  25. 25.
    Gorgun FM, Zhuo M, Singh S, Englander EW (2014) Neuroglobin mitigates mitochondrial impairments induced by acute inhalation of combustion smoke in the mouse brain. Inhal Toxicol 26:361–369CrossRefGoogle Scholar
  26. 26.
    Gorgun MF, Zhuo M, Cortez I, Dineley KT, Englander EW (2017) Acute inhalation of combustion smoke triggers neuroinflammation and persistent anxiety-like behavior in the mouse. Inhal Toxicol 29:598–610CrossRefGoogle Scholar
  27. 27.
    Chen L, Lee HM, Greeley GH Jr, Englander EW (2007) Accumulation of oxidatively generated DNA damage in the brain: a mechanism of neurotoxicity. Free Radic Biol Med 42:385–393CrossRefGoogle Scholar
  28. 28.
    Hammelrath L, Skokic S, Khmelinskii A, Hess A, van der Knaap N, Staring M, Lelieveldt BPF, Wiedermann D, Hoehn M (2016) Morphological maturation of the mouse brain: an in vivo MRI and histology investigation. Neuroimage 125:144–152CrossRefGoogle Scholar
  29. 29.
    Tu TW, Kim JH, Yin FQ, Jakeman LB, Song SK (2013) The impact of myelination on axon sparing and locomotor function recovery in spinal cord injury assessed using diffusion tensor imaging. NMR Biomed 26:1484–1495CrossRefGoogle Scholar
  30. 30.
    Zhang H, Yan G, Xu H, Fang Z, Zhang J, Zhang J, Wu R, Kong J, Huang Q (2016) The recovery trajectory of adolescent social defeat stress-induced behavioral, (1)H-MRS metabolites and myelin changes in Balb/c mice. Sci Rep 6:27906CrossRefGoogle Scholar
  31. 31.
    Zhuo M, Gorgun MF, Englander EW (2018) Translesion synthesis DNA polymerase kappa is indispensable for DNA repair synthesis in cisplatin exposed dorsal root ganglion neurons. Mol Neurobiol 55:2506–2515CrossRefGoogle Scholar
  32. 32.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  33. 33.
    Jao CY, Salic A (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci U S A 105:15779-15784CrossRefGoogle Scholar
  34. 34.
    Dineley KT, Hogan D, Zhang WR, Taglialatela G (2007) Acute inhibition of calcineurin restores associative learning and memory in Tg2576 APP transgenic mice. Neurobiol Learn Mem 88:217–224CrossRefGoogle Scholar
  35. 35.
    Dineley KT, Xia X, Bui D, Sweatt JD, Zheng H (2002) Accelerated plaque accumulation, associative learning deficits, and up-regulation of alpha 7 nicotinic receptor protein in transgenic mice co-expressing mutant human presenilin 1 and amyloid precursor proteins. J Biol Chem 277:22768–22780CrossRefGoogle Scholar
  36. 36.
    Taglialatela G, Hogan D, Zhang WR, Dineley KT (2009) Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition. Behav Brain Res 200:95–99CrossRefGoogle Scholar
  37. 37.
    Crawley JN, Paylor R (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Behav 31:197–211CrossRefGoogle Scholar
  38. 38.
    Bevins RA, Besheer J (2006) Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study 'recognition memory'. Nat Protoc 1:1306–1311CrossRefGoogle Scholar
  39. 39.
    Hernandez CM, Kayed R, Zheng H, Sweatt JD, Dineley KT (2010) Loss of alpha7 nicotinic receptors enhances beta-amyloid oligomer accumulation, exacerbating early-stage cognitive decline and septohippocampal pathology in a mouse model of Alzheimer's disease. J Neurosci 30:2442–2453CrossRefGoogle Scholar
  40. 40.
    Leger M, Quiedeville A, Bouet V, Haelewyn B, Boulouard M, Schumann-Bard P, Freret T (2013) Object recognition test in mice. Nat Protoc 8:2531–2537CrossRefGoogle Scholar
  41. 41.
    Pavlopoulos E, Jones S, Kosmidis S, Close M, Kim C, Kovalerchik O, Small SA, Kandel ER (2013) Molecular mechanism for age-related memory loss: the histone-binding protein RbAp48. Sci Transl Med 5:200ra115CrossRefGoogle Scholar
  42. 42.
    Prokop S, Miller KR, Heppner FL (2013) Microglia actions in Alzheimer's disease. Acta Neuropathol 126:461–477CrossRefGoogle Scholar
  43. 43.
    Xu H, Zhang SL, Tan GW, Zhu HW, Huang CQ, Zhang FF, Wang ZX (2012) Reactive gliosis and neuroinflammation in rats with communicating hydrocephalus. Neuroscience 218:317–325CrossRefGoogle Scholar
  44. 44.
    Schregel K, Wuerfel E, Garteiser P, Gemeinhardt I, Prozorovski T, Aktas O, Merz H, Petersen D, Wuerfel J, Sinkus R (2012) Demyelination reduces brain parenchymal stiffness quantified in vivo by magnetic resonance elastography. Proc Natl Acad Sci USA 109:6650–6655CrossRefGoogle Scholar
  45. 45.
    Becher B, Spath S, Goverman J (2017) Cytokine networks in neuroinflammation. Nat Rev Immunol 17:49–59CrossRefGoogle Scholar
  46. 46.
    Moreno-Jimenez EP, Flor-Garcia M, Terreros-Roncal J, Rabano A, Cafini F, Pallas-Bazarra N, Avila J, Llorens-Martin M (2019) Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease. Nat Med 25:554–560CrossRefGoogle Scholar
  47. 47.
    Zywitza V, Misios A, Bunatyan L, Willnow TE, Rajewsky N (2018) Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects impairing adult neurogenesis. Cell Rep 25:2457–2469CrossRefGoogle Scholar
  48. 48.
    Pieribone VA, Porton B, Rendon B, Feng J, Greengard P, Kao HT (2002) Expression of synapsin III in nerve terminals and neurogenic regions of the adult brain. J Comp Neurol 454:105–114CrossRefGoogle Scholar
  49. 49.
    Alarie Y (2002) Toxicity of fire smoke. Crit Rev Toxicol 32:259–289CrossRefGoogle Scholar
  50. 50.
    Smith SM, Stuhmiller JH, Januszkiewicz AJ (1996) Evaluation of lethality estimates for combustion gases in military scenarios. Toxicology 115:157–165CrossRefGoogle Scholar
  51. 51.
    Stuhmiller JH, Long DW, Stuhmiller LM (2006) An internal dose model of incapacitation and lethality risk from inhalation of fire gases. Inhal Toxicol 18:347–364CrossRefGoogle Scholar
  52. 52.
    Baud F, Boukobza M, Borron SW (2011) Cyanide: an unreported cause of neurological complications following smoke inhalation. BMJ Case Rep. Google Scholar
  53. 53.
    Dries DJ, Endorf FW (2013) Inhalation injury: epidemiology, pathology, treatment strategies. Scand J Trauma Resusc Emerg Med 21:31CrossRefGoogle Scholar
  54. 54.
    Geldner G, Koch EM, Gottwald-Hostalek U, Baud F, Burillo G, Fauville JP, Levi F, Locatelli C, Zilker T (2013) Report on a study of fires with smoke gas development: determination of blood cyanide levels, clinical signs and laboratory values in victims. Anaesthesist 62:609–616CrossRefGoogle Scholar
  55. 55.
    Huang CC, Chung MH, Weng SF, Chien CC, Lin SJ, Lin HJ, Guo HR, Su SB, Hsu CC, Juan CW (2014) Long-term prognosis of patients with carbon monoxide poisoning: a nationwide cohort study. PLoS ONE 9:e105503CrossRefGoogle Scholar
  56. 56.
    Mizuno Y, Sakurai Y, Sugimoto I, Ichinose K, Ishihara S, Sanjo N, Mizusawa H, Mannen T (2014) Delayed leukoencephalopathy after carbon monoxide poisoning presenting as subacute dementia. Intern Med 53:1441–1445CrossRefGoogle Scholar
  57. 57.
    Pages B, Planton M, Buys S, Lemesle B, Birmes P, Barbeau EJ, Maziero S, Cordier L, Cabot C, Puel M, Genestal M, Chollet F, Pariente J (2014) Neuropsychological outcome after carbon monoxide exposure following a storm: a case-control study. BMC Neurol 14:153CrossRefGoogle Scholar
  58. 58.
    Pang L, Wang HL, Wang ZH, Wu Y, Dong N, Xu DH, Wang DW, Xu H, Zhang N (2014) Plasma copeptin as a predictor of intoxication severity and delayed neurological sequelae in acute carbon monoxide poisoning. Peptides 59:89–93CrossRefGoogle Scholar
  59. 59.
    Tsai CF, Yip PK, Chen SY, Lin JC, Yeh ZT, Kung LY, Wang CY, Fan YM (2014) The impacts of acute carbon monoxide poisoning on the brain: longitudinal clinical and 99mTc ethyl cysteinate brain SPECT characterization of patients with persistent and delayed neurological sequelae. Clin Neurol Neurosurg 119:21–27CrossRefGoogle Scholar
  60. 60.
    Yeh ZT, Tsai CF, Yip PK, Lo CY, Peng SM, Chen SY, Kung LY (2014) Neuropsychological performance in patients with carbon monoxide poisoning. Appl Neuropsychol Adult 21:278–287CrossRefGoogle Scholar
  61. 61.
    Anseeuw K, Delvau N, Burillo-Putze G, De Iaco F, Geldner G, Holmstrom P, Lambert Y, Sabbe M (2013) Cyanide poisoning by fire smoke inhalation: a European expert consensus. Eur J Emerg Med 20:2–9CrossRefGoogle Scholar
  62. 62.
    Antonio AC, Castro PS, Freire LO (2013) Smoke inhalation injury during enclosed-space fires: an update. J Bras Pneumol 39:373–381CrossRefGoogle Scholar
  63. 63.
    Lawson-Smith P, Jansen EC, Hyldegaard O (2011) Cyanide intoxication as part of smoke inhalation–a review on diagnosis and treatment from the emergency perspective. Scand J Trauma Resusc Emerg Med 19:14CrossRefGoogle Scholar
  64. 64.
    Chen F, Lu J, Chen F, Lin Z, Lin Y, Yu L, Su X, Yao P, Cai B, Kang D (2018) Recombinant neuroglobin ameliorates early brain injury after subarachnoid hemorrhage via inhibiting the activation of mitochondria apoptotic pathway. Neurochem Int 112:219–226CrossRefGoogle Scholar
  65. 65.
    Xiong XX, Pan F, Chen RQ, Hu DX, Qiu XY, Li CY, Xie XQ, Tian B, Chen XQ (2018) Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal. Cell Death Dis 9:163CrossRefGoogle Scholar
  66. 66.
    Zhang B, Ji X, Zhang S, Ren H, Wang M, Guo C, Li Y (2013) Heminmediated neuroglobin induction exerts neuroprotection following ischemic brain injury through PI3K/Akt signaling. Mol Med Rep 8:681–685CrossRefGoogle Scholar
  67. 67.
    Chen LM, Xiong YS, Kong FL, Qu M, Wang Q, Chen XQ, Wang JZ, Zhu LQ (2012) Neuroglobin attenuates Alzheimer-like tau hyperphosphorylation by activating Akt signaling. J Neurochem 120:157–164CrossRefGoogle Scholar
  68. 68.
    Khan AA, Mao XO, Banwait S, Jin K, Greenberg DA (2007) Neuroglobin attenuates beta-amyloid neurotoxicity in vitro and transgenic Alzheimer phenotype in vivo. Proc Natl Acad Sci USA 104:19114–19119CrossRefGoogle Scholar
  69. 69.
    Liu N, Yu Z, Xun Y, Shu P, Yue Y, Yuan S, Jiang Y, Huang Z, Yang X, Feng X, Xiang S, Wang X (2018) Amyloid-beta25-35 upregulates endogenous neuroprotectant neuroglobin via NFkappaB activation in vitro. J Alzheimers Dis 64:1163–1174CrossRefGoogle Scholar
  70. 70.
    Zara S, De Colli M, Rapino M, Pacella S, Nasuti C, Sozio P, Di Stefano A, Cataldi A (2013) Ibuprofen and lipoic acid conjugate neuroprotective activity is mediated by Ngb/Akt intracellular signaling pathway in Alzheimer's disease rat model. Gerontology 59:250–260CrossRefGoogle Scholar
  71. 71.
    Nicolis S, Monzani E, Pezzella A, Ascenzi P, Sbardella D, Casella L (2013) Neuroglobin modification by reactive quinone species. Chem Res Toxicol 26:1821–1831CrossRefGoogle Scholar
  72. 72.
    Watanabe S, Takahashi N, Uchida H, Wakasugi K (2012) Human neuroglobin functions as an oxidative stress-responsive sensor for neuroprotection. J Biol Chem 287:30128–30138CrossRefGoogle Scholar
  73. 73.
    Watanabe S, Wakasugi K (2008) Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity. Biochem Biophys Res Commun 369:695–700CrossRefGoogle Scholar
  74. 74.
    Cai B, Li W, Mao X, Winters A, Ryou MG, Liu R, Greenberg DA, Wang N, Jin K, Yang SH (2015) Neuroglobin overexpression inhibits AMPK signaling and promotes cell anabolism. Mol Neurobiol 53:1254–1265CrossRefGoogle Scholar
  75. 75.
    Yan W (2016) Carbon monoxide, the silent killer, may have met its match. Science 354:1215CrossRefGoogle Scholar
  76. 76.
    Zou YY, Kan EM, Cao Q, Lu J, Ling EA (2013) Combustion smoke-induced inflammation in the cerebellum and hippocampus of adult rats. Neuropathol Appl Neurobiol 39:531–552CrossRefGoogle Scholar
  77. 77.
    Warburton EC, Brown MW (2015) Neural circuitry for rat recognition memory. Behav Brain Res 285:131–139CrossRefGoogle Scholar
  78. 78.
    Van Leuven W, Van Dam D, Moens L, De Deyn PP, Dewilde S (2013) A behavioural study of neuroglobin-overexpressing mice under normoxic and hypoxic conditions. Biochim Biophys Acta 1834:1764–1771CrossRefGoogle Scholar
  79. 79.
    Lipovsek M, Grubb MS (2019) Boosting adult neurogenesis to enhance sensory performance. EMBO J 38:e101589CrossRefGoogle Scholar
  80. 80.
    Goncalves JT, Schafer ST, Gage FH (2016) Adult neurogenesis in the hippocampus: from stem cells to behavior. Cell 167:897–914CrossRefGoogle Scholar
  81. 81.
    Yu Z, Cheng C, Liu Y, Liu N, Lo EH, Wang X (2018) Neuroglobin promotes neurogenesis through Wnt signaling pathway. Cell Death Dis 9:945CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Surgery, Medical BranchUniversity of TexasGalvestonUSA
  2. 2.Department of NeurologyUniversity of Texas Medical BranchGalvestonUSA
  3. 3.Mitchell Center for Neurodegenerative DiseasesUniversity of Texas Medical BranchGalvestonUSA
  4. 4.Center for Addiction ResearchUniversity of Texas Medical BranchGalvestonUSA
  5. 5.Shriners Hospitals for ChildrenGalvestonUSA

Personalised recommendations