, Volume 15, Issue 1, pp 216–232 | Cite as

iTRAQ-Based Quantitative Proteomics Reveals the New Evidence Base for Traumatic Brain Injury Treated with Targeted Temperature Management

Original Article


This study aimed to investigate the effects of targeted temperature management (TTM) modulation on traumatic brain injury (TBI) and the involved mechanisms using quantitative proteomics technology. SH-SY5Y and HT-22 cells were subjected to moderate stretch injury using the cell injury controller (CIC), followed by incubation at TTM (mild hypothermia, 32°C), or normothermia (37°C). The real-time morphological changes, cell cycle phase distribution, death, and cell viability were evaluated. Moderate TBI was produced by the controlled cortical impactor (CCI), and the effects of TTM on the neurological damage, neurodegeneration, cerebrovascular histopathology, and behavioral outcome were determined in vivo. Results showed that TTM treatment prevented TBI-induced neuronal necrosis in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, reduced cortical lesion volume and neuronal loss, attenuated cerebrovascular histopathological damage, brain edema, and improved behavioral outcome. Using an iTRAQ proteomics approach, proteins that were significantly associated with TTM in experimental TBI were identified. Importantly, changes in four candidate molecules (plasminogen [PLG], antithrombin III [AT III], fibrinogen gamma chain [FGG], transthyretin [TTR]) were verified using TBI rat brain tissues and TBI human cerebrospinal fluid (CSF) samples. This study is one of the first to investigate the neuroprotective effects of TTM on the proteome of human and experimental models of TBI, providing an overall landscape of the TBI brain proteome and a scientific foundation for further assessment of candidate molecules associated with TTM for the promotion of reparative strategies post-TBI.


Targeted temperature management Traumatic brain injury Proteomics Isobaric tags for relative and absolute quantitation Cerebrospinal fluid 



This work was supported by grants from the National Natural Science Foundation of China (31200809), Science & Technology Program of Tianjin, China (15ZXLCSY00040), and Technology Research Projects (AWS15J001).

Required Author Forms

Disclosure forms provided by the authors are available with the online version of this article.

Supplementary material

13311_2017_591_MOESM1_ESM.xlsx (707 kb)
Table S1 (XLSX 706 kb)
13311_2017_591_MOESM2_ESM.xlsx (97 kb)
Table S2 (XLSX 97 kb)
13311_2017_591_MOESM3_ESM.xlsx (18 kb)
Table S3 (XLSX 17 kb)
13311_2017_591_MOESM4_ESM.docx (57 kb)
Fig. S1 (DOCX 56 kb)
13311_2017_591_MOESM5_ESM.pdf (1.2 mb)
ESM 1 (PDF 1224 kb)


  1. 1.
    Manley GT, Maas AI. Traumatic brain injury: an international knowledge-based approach. JAMA 2013;310:473-474.CrossRefPubMedGoogle Scholar
  2. 2.
    Jiang JY. Head trauma in China. Injury 2013;44:1453-1457.CrossRefPubMedGoogle Scholar
  3. 3.
    Corrigan JD, Selassie AW, Orman JA. The epidemiology of traumatic brain injury. J Head Trauma Rehabil 2010;25:72-80.CrossRefPubMedGoogle Scholar
  4. 4.
    Nunnally ME, Jaeschke R, Bellingan GJ et al. Targeted temperature management in critical care: a report and recommendations from five professional societies. Crit Care Med 2011;39:1113-1125.CrossRefPubMedGoogle Scholar
  5. 5.
    Zhao CC, Wang CF, Li WP et al. Mild hypothermia promotes pericontusion neuronal sprouting via suppressing suppressor of cytokine signaling 3 expression after moderate traumatic brain injury. J Neurotrauma 2017;34:1636-1644.CrossRefPubMedGoogle Scholar
  6. 6.
    Truettner JS, Bramlett HM, Dietrich WD. Posttraumatic therapeutic hypothermia alters microglial and macrophage polarization toward a beneficial phenotype. J Cereb Blood Flow Metab 2016;271678X16680003.Google Scholar
  7. 7.
    Zhang HB, Cheng SX, Tu Y, Zhang S, Hou SK, Yang Z. Protective effect of mild-induced hypothermia against moderate traumatic brain injury in rats involved in necroptotic and apoptotic pathways. Brain Inj 2017;31:406-415.CrossRefPubMedGoogle Scholar
  8. 8.
    Liu T, Zhao DX, Cui H et al. Therapeutic hypothermia attenuates tissue damage and cytokine expression after traumatic brain injury by inhibiting necroptosis in the rat. Sci Rep 2016;6:24547.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Diller KR, Zhu L. Hypothermia therapy for brain injury. Annu Rev Biomed Eng 2009;11:135-162.CrossRefPubMedGoogle Scholar
  10. 10.
    Gibbons H, Sato TA, Dragunow M. Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells. Brain Res Mol Brain Res 2003;110:63-75.CrossRefPubMedGoogle Scholar
  11. 11.
    Amin FU, Shah SA, Kim MO. Vanillic acid attenuates Abeta1-42-induced oxidative stress and cognitive impairment in mice. Sci Rep 2017;7:40753.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Urbano LA, Oddo M. Therapeutic hypothermia for traumatic brain injury. Curr Neurol Neurosci Rep 2012;12:580-591.CrossRefPubMedGoogle Scholar
  13. 13.
    Andriessen TM, Jacobs B, Vos PE. Clinical characteristics and pathophysiological mechanisms of focal and diffuse traumatic brain injury. J Cell Mol Med 2010;14:2381-2392.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Clifton GL, Valadka A, Zygun D et al. Very early hypothermia induction in patients with severe brain injury (the National Acute Brain Injury Study: Hypothermia II): a randomised trial. Lancet Neurol 2011;10:131-139.CrossRefPubMedGoogle Scholar
  15. 15.
    Ottens AK, Bustamante L, Golden EC et al. Neuroproteomics: a biochemical means to discriminate the extent and modality of brain injury. J Neurotrauma 2010;27:1837-1852.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chahrour O, Cobice D, Malone J. Stable isotope labelling methods in mass spectrometry-based quantitative proteomics. J Pharm Biomed Anal 2015;113:2-20.CrossRefPubMedGoogle Scholar
  17. 17.
    Morrison B, 3rd, Elkin BS, Dolle JP, Yarmush ML. In vitro models of traumatic brain injury. Annu Rev Biomed Eng 2011;13:91-126.CrossRefPubMedGoogle Scholar
  18. 18.
    Thelin EP, Nelson DW, Bellander BM. A review of the clinical utility of serum S100B protein levels in the assessment of traumatic brain injury. Acta Neurochir (Wien) 2017;159:209-225.CrossRefGoogle Scholar
  19. 19.
    Fujita M, Oda Y, Yamashita S et al. Early-stage hyperoxia is associated with favorable neurological outcomes and survival after severe traumatic brain injury: a post-hoc analysis of the brain hypothermia study. J Neurotrauma 2017;19Google Scholar
  20. 20.
    Andrews PJ, Sinclair HL, Rodriguez A et al. Hypothermia for intracranial hypertension after traumatic brain injury. N Engl J Med 2015;373:2403-2412.CrossRefPubMedGoogle Scholar
  21. 21.
    Cheng SX, Zhang S, Sun HT, Tu Y. Effects of mild hypothermia treatment on rat hippocampal beta-amyloid expression following traumatic brain injury. Ther Hypothermia Temp Manag 2013;3:132-139.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Loov C, Nadadhur AG, Hillered L, Clausen F, Erlandsson A. Extracellular ezrin: a novel biomarker for traumatic brain injury. J Neurotrauma 2015;32:244-251.CrossRefPubMedGoogle Scholar
  23. 23.
    Silasi G, Colbourne F. Unilateral brain hypothermia as a method to examine efficacy and mechanisms of neuroprotection against global ischemia. Ther Hypothermia Temp Manag 2011;1:87-94.CrossRefPubMedGoogle Scholar
  24. 24.
    Jin Y, Lei J, Lin Y, Gao GY, Jiang JY. Autophagy inhibitor 3-MA weakens neuroprotective effects of posttraumatic brain injury moderate hypothermia. World Neurosurg 2016;88:433-446.CrossRefPubMedGoogle Scholar
  25. 25.
    Marion DW, Penrod LE, Kelsey SF et al. Treatment of traumatic brain injury with moderate hypothermia. N Engl J Med 1997;336:540-546.CrossRefPubMedGoogle Scholar
  26. 26.
    Hsieh CL, Niemi EC, Wang SH et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 2014;31:1677-1688.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bareyre F, Wahl F, McIntosh TK, Stutzmann JM. Time course of cerebral edema after traumatic brain injury in rats: effects of riluzole and mannitol. J Neurotrauma 1997;14:839-849.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhao J, Pati S, Redell JB, Zhang M, Moore AN, Dash PK. Caffeic acid phenethyl ester protects blood-brain barrier integrity and reduces contusion volume in rodent models of traumatic brain injury. J Neurotrauma 2012;29:1209-1218.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Anderson KJ, Fugaccia I, Scheff SW. Fluoro-jade B stains quiescent and reactive astrocytes in the rodent spinal cord. J Neurotrauma 2003;20:1223-1231.CrossRefPubMedGoogle Scholar
  30. 30.
    Gold EM, Su D, Lopez-Velazquez L et al. Functional assessment of long-term deficits in rodent models of traumatic brain injury. Regen Med 2013;8:483-516.CrossRefPubMedGoogle Scholar
  31. 31.
    Tu Y, Chen C, Sun HT et al. Combination of temperature-sensitive stem cells and mild hypothermia: a new potential therapy for severe traumatic brain injury. J Neurotrauma 2012;29:2393-2403.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Zhai L, Chang C, Li N et al. Systematic research on the pretreatment of peptides for quantitative proteomics using a C(1)(8) microcolumn. Proteomics 2013;13:2229-2237.CrossRefPubMedGoogle Scholar
  33. 33.
    Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4:44-57.CrossRefPubMedGoogle Scholar
  34. 34.
    Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009;37:1-13.CrossRefPubMedGoogle Scholar
  35. 35.
    Lei J, Gao G, Mao Q et al. Rationale, methodology, and implementation of a nationwide multicenter randomized controlled trial of long-term mild hypothermia for severe traumatic brain injury (the LTH-1 trial). Contemp Clin Trials 2015;40:9-14.CrossRefPubMedGoogle Scholar
  36. 36.
    Jin X, Xu Z, Cao J, et al. Proteomics analysis of human placenta reveals glutathione metabolism dysfunction as the underlying pathogenesis for preeclampsia. BBA 2017;1865:1207-1214.Google Scholar
  37. 37.
    Jin G, Liu B, You Z, et al. Development of a novel neuroprotective strategy: combined treatment with hypothermia and valproic acid improves survival in hypoxic hippocampal cells. Surgery 2014;156:221-228.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    de Oliveira Manoel AL, Neto AC, Veigas PV, Rizoli S. Traumatic brain injury associated coagulopathy. Neurocrit Care 2015;22:34-44.CrossRefPubMedGoogle Scholar
  39. 39.
    Benoit CE, Rowe WB, Menard C, Sarret P, Quirion R. Genomic and proteomic strategies to identify novel targets potentially involved in learning and memory. Trends Pharmacol Sci 2011;32:43-52.CrossRefPubMedGoogle Scholar
  40. 40.
    Bregy A, Nixon R, Lotocki G et al. Posttraumatic hypothermia increases doublecortin expressing neurons in the dentate gyrus after traumatic brain injury in the rat. Exp Neurol 2012;233:821-828.CrossRefPubMedGoogle Scholar
  41. 41.
    Huang T, Solano J, He D, Loutfi M, Dietrich WD, Kuluz JW. Traumatic injury activates MAP kinases in astrocytes: mechanisms of hypothermia and hyperthermia. J Neurotrauma 2009;26:1535-1545.CrossRefPubMedGoogle Scholar
  42. 42.
    Oda Y, Gao G, Wei EP, Povlishock JT. Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood-brain barrier dysfunction after traumatic brain injury in rat. J Cereb Blood Flow Metab 2011;31:1143-1154.CrossRefPubMedGoogle Scholar
  43. 43.
    Hatic H, Kane MJ, Saykally JN, Citron BA. Modulation of transcription factor Nrf2 in an in vitro model of traumatic brain injury. J Neurotrauma 2012;29:1188-1196.CrossRefPubMedGoogle Scholar
  44. 44.
    Huang L, Coats JS, Mohd-Yusof A et al. Tissue vulnerability is increased following repetitive mild traumatic brain injury in the rat. Brain Res 2013;1499:109-120.CrossRefPubMedGoogle Scholar
  45. 45.
    Gu X, Wei ZZ, Espinera A et al. Pharmacologically induced hypothermia attenuates traumatic brain injury in neonatal rats. Exp Neurol 2015;267:135-142.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Lee JH, Wei L, Gu X, Wei Z, Dix TA, Yu SP. Therapeutic effects of pharmacologically induced hypothermia against traumatic brain injury in mice. J Neurotrauma 2014;31:1417-1430.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Lotocki G, de Rivero Vaccari JP, Perez ER et al. Alterations in blood-brain barrier permeability to large and small molecules and leukocyte accumulation after traumatic brain injury: effects of post-traumatic hypothermia. J Neurotrauma 2009;26:1123-1134.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Doll H, Maegele M, Bohl J et al. Pharyngeal selective brain cooling is associated with reduced CNS cortical lesion after experimental traumatic brain injury in rats. J Neurotrauma 2010;27:2245-2254.CrossRefPubMedGoogle Scholar
  49. 49.
    Fujita M, Wei EP, Povlishock JT. Effects of hypothermia on cerebral autoregulatory vascular responses in two rodent models of traumatic brain injury. J Neurotrauma 2012;29:1491-1498.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Miyauchi T, Wei EP, Povlishock JT. Evidence for the therapeutic efficacy of either mild hypothermia or oxygen radical scavengers after repetitive mild traumatic brain injury. J Neurotrauma 2014;31:773-781.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Szczygielski J, Muller A, Mautes AE et al. Selective brain hypothermia mitigates brain damage and improves neurological outcome after post-traumatic decompressive craniectomy in mice. J Neurotrauma 2017;34:1623-1635.CrossRefPubMedGoogle Scholar
  52. 52.
    Feala JD, Abdulhameed MD, Yu C et al. Systems biology approaches for discovering biomarkers for traumatic brain injury. J Neurotrauma 2013;30:1101-1116.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Kell DB, Oliver SG. Here is the evidence, now what is the hypothesis? The complementary roles of inductive and hypothesis-driven science in the post-genomic era. Bioessays 2004;26:99-105.CrossRefPubMedGoogle Scholar
  54. 54.
    Garland P, Broom LJ, Quraishe S et al. Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton. PLOS ONE 2012;7:e47552.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Salehi A, Zhang JH, Obenaus A. Response of the cerebral vasculature following traumatic brain injury. J Cereb Blood Flow Metab 2017;37:2320-2339.CrossRefPubMedGoogle Scholar
  56. 56.
    Thelin EP, Frostell A, Mulder J et al. Lesion size is exacerbated in hypoxic rats whereas hypoxia-inducible factor-1 alpha and vascular endothelial growth factor increase in injured normoxic rats: a prospective cohort study of secondary hypoxia in focal traumatic brain injury. Front Neurol 2016;7:23.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Thelin EP, Jeppsson E, Frostell A et al. Utility of neuron-specific enolase in traumatic brain injury; relations to S100B levels, outcome, and extracranial injury severity. Crit Care 2016;20:285.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Thelin EP, Just D, Frostell A et al. Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers. Behav Brain Res 2016.
  59. 59.
    Yang DB, Yu WH, Dong XQ et al. Serum macrophage migration inhibitory factor concentrations correlate with prognosis of traumatic brain injury. Clin Chim Acta 2017;469:99-104.CrossRefPubMedGoogle Scholar
  60. 60.
    Sharma R, Rosenberg A, Bennett ER, Laskowitz DT, Acheson SK. A blood-based biomarker panel to risk-stratify mild traumatic brain injury. PLOS ONE 2017;12:e0173798.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The American Society for Experimental NeuroTherapeutics, Inc. 2017

Authors and Affiliations

  1. 1.Tianjin Key Laboratory of Neurotrauma Repair, Institute of Traumatic Brain Injury and Neuroscience, Center for Neurology and Neurosurgery of Affiliated HospitalLogistics University of Chinese People’s Armed Police Force (PAP)TianjinChina
  2. 2.Central Laboratory of Logistics University of Chinese People’s Armed Police Force (PAP)TianjinChina

Personalised recommendations