Neurochemical Research

, Volume 43, Issue 9, pp 1779–1790 | Cite as

Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats

  • Ya-Jie Huang
  • Yu-Jia Yuan
  • Yi-Xian Liu
  • Meng-Yue Zhang
  • Jing-Ge ZhangEmail author
  • Tian-Ci Wang
  • Li-Nan Zhang
  • Yu-Yan Hu
  • Li Li
  • Xiao-Hui Xian
  • Jie Qi
  • Min ZhangEmail author
Original Paper


Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH + sham group, ischemia group and IH + ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (PB = 404 mmHg, PO2 = 84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, l-NAME + ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of l-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.


Intermittent hypobaric hypoxia Global brain ischemia Nitric oxide synthase Nitric oxide Hippocampal CA1 subfield 



Bilateral common carotid arteries


Delayed neuronal death




Histological grade


Intermittent hypobaric hypoxia


Inducible nitric oxide synthase


N-nitro-l-arginine methyl ester


Neuronal density


Nitric oxide


Nitric oxide synthase


Neuronal nitric oxide synthase


Phosphate-buffered saline


Standard devariance



The present study was supported by the National Natural Science Foundation of China (Nos. 81771253, 31271149 and 81271454), Natural Science Foundation of Hebei Province, China (No. H2015206492) and College Students’ innovation experiment program (USIP201503A).

Compliance with Ethical Standards

Conflict of interest

We declare no conflicts of interest in this study.

Ethical Approval

All procedures performed in studies involving animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Care of Hebei Medical University.


  1. 1.
    Wu Q, Yu KX, Ma QS, Liu YN (2015) Effects of intermittent hypobaric hypoxia preconditioning on the expression of neuroglobin and Bcl-2 in the rat hippocampal CA1 area following ischemia-reperfusion. Genet Mol Res 14(3):10799–10807CrossRefPubMedGoogle Scholar
  2. 2.
    Wang J, Zhang S, Ma H, Yang S, Liu Z, Wu X, Wang S, Zhang Y, Liu Y (2017) Chronic intermittent hypobaric hypoxia pretreatment ameliorates ischemia-induced cognitive dysfunction through activation of ERK1/2-creb-bdnf pathway in anesthetized mice. Neurochem Res 42(2):501–512CrossRefPubMedGoogle Scholar
  3. 3.
    O’Dell TJ, Hawkins RD, Kandel ER, Arancio O (1991) Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc Natl Acad Sci USA 88(24):11285–11289CrossRefPubMedGoogle Scholar
  4. 4.
    Holán V, Krulová M, Zajícová A, Pindjáková J (2002) Nitric oxide as a regulatory and effector molecule in the immune system. Mol Immunol 38(12–13):989–995CrossRefPubMedGoogle Scholar
  5. 5.
    Banuls C, Rocha M, Rovirallopis S, Falcon R, Castello R, Herance JR, Polo M, Blas-Garcia A, Hernandez-Mijares A, Victor VM (2014) The pivotal role of nitric oxide: effects on the nervous and immune systems. Curr Pharm Des 20(29):4679–4689CrossRefPubMedGoogle Scholar
  6. 6.
    Phattanarudee S, Towiwat P, Maher TJ, Ally A (2013) Effects of medullary administration of a nitric oxide precursor on cardiovascular responses and neurotransmission during static exercise following ischemic stroke. Can J Physiol Pharmacol 91(7):510CrossRefPubMedGoogle Scholar
  7. 7.
    Koylu EO, Kanit L, Taskiran D, Dagci T, Balkan B, Pogun S (2005) Effects of nitric oxide synthase inhibition on spatial discrimination learning and central DA2 and Mach receptors. Pharmacol Biochem Behav 81(1):32–40CrossRefPubMedGoogle Scholar
  8. 8.
    Zhang Y, Li NZ, Yang Z (2010) Perinatal food restriction impaired spatial learning and memory behavior and decreased the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring. Toxicol Lett 193(2):167–172CrossRefPubMedGoogle Scholar
  9. 9.
    Terpolilli NA, Moskowitz MA, Plesnila N (2012) Nitric oxide: considerations for the treatment of ischemic stroke. J Cereb Blood Flow Metab 32(7):1332–1346CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Shapshak P (2012) Single nucleotide polymorphisms (SNPs) for genome wide association studies (GWAS) and molecule of the month Nitric Oxide Synthase, multiple interactive pathways for three similar genes, Nitric Oxide Synthase-1,-2,-3(NOS-1,-2,-3). Bioinformation 8(11):496–497CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Poulos TL, Li H (2017) Nitric oxide synthase and structure-based inhibitor design. Nitric Oxide 63:68–77CrossRefPubMedGoogle Scholar
  12. 12.
    Wang M, Qi DS, Zhou C, Han D, Li PP, Zhang F, Zhou XY, Han M, Di JH, Ye JS, Yu HM, Song YJ, Zhang GY (2016) Ischemic preconditioning protects the brain against injury via inhibiting CaMKII-nNOS signaling pathway. Brain Res 1634:140–149CrossRefPubMedGoogle Scholar
  13. 13.
    Rocha-Ferreira E, Rudge B, Hughes MP, Rahim AA, Hristova M, Robertson NJ (2016) Immediate remote ischemic postconditioning reduces brain nitrotyrosine formation in a piglet asphyxia model. Oxid Med Cell Longev 2016(1):1–11CrossRefGoogle Scholar
  14. 14.
    Iadecola C, Xu X, Zhang F, El-Fakahany EE, Ross ME (1995) Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia. J Cereb Blood Flow Metab 15(1):52–59CrossRefPubMedGoogle Scholar
  15. 15.
    Ono K, Suzuki H, Sawada M (2010) Delayed neural damage is induced by iNOS-expressing microglia in a brain injury model. Neurosci Lett 473(2):146–150CrossRefPubMedGoogle Scholar
  16. 16.
    Yuan F, Guo Z, Xu Y, Wang X, Bu HM, Zhong N, Zhang Y, Zhou ZN (2008) Comparison of the effects of chronic intermittent hypobaric hypoxia and continuous hypobaric hypoxia on hemodynamics in rats. Sheng Li Xue Bao 60:687–694PubMedGoogle Scholar
  17. 17.
    Shi M, Cui F, Liu AJ, Li J, Ma HJ, Cheng M, Yang J, Zhang Y (2011) Protection of chronic intermittent hypobaric hypoxia against collagen-induced arthritis in rat through increasing apoptosis. Sheng Li Xue Bao 63:115–123PubMedGoogle Scholar
  18. 18.
    Kato H, Liu Y, Araki T, Kogure K (1991) Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res 553(2):238–242CrossRefPubMedGoogle Scholar
  19. 19.
    Gong SJ, Chen LY, Zhang M, Gong JX, Ma YX, Zhang JM, Wang YJ, Hu YY, Sun XC, Li WB, Zhang Y (2012) Intermittent hypobaric hypoxia preconditioning induced brain ischemic tolerance by up-regulating glial glutamate transporter-1 in rats. Neurochem Res 37(3):527–537CrossRefPubMedGoogle Scholar
  20. 20.
    Pulsinelli WA, Brierley JB (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10(3):267–272CrossRefPubMedGoogle Scholar
  21. 21.
    Liu HQ, Li WB, Li QJ, Zhang M, Sun XC, Feng RF, Xian XH, Qi J, Zhao HG (2006) Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem Res 31(7):967–974CrossRefPubMedGoogle Scholar
  22. 22.
    Zhang M, Li WB, Geng JX, Li QJ, Sun XC, Xian XH, Qi J, Li SQ (2007) The up-regulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats. J Cereb Blood Flow Metab 27(7):1352–1368CrossRefPubMedGoogle Scholar
  23. 23.
    Lehotský J, Tothová B, Kovalská M, Dobrota D, Beňová A, Kalenská D, Kaplán P (2016) Role of homocysteine in the ischemic stroke and development of ischemic tolerance. Front Neurosci 10:538CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Liu ZJ, Chen C, Li XR, Ran YY, Xu T, Zhang Y, Geng XK, Zhang Y, Du HS, Leak RK, Ji XM, Hu XM (2016) Remote ischemic preconditioning-mediated neuroprotection against stroke is associated with significant alterations in peripheral immune responses. CNS Neurosci Ther 22(1):43–52CrossRefPubMedGoogle Scholar
  25. 25.
    Schmidtkastner R, Freund TF (1991) Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 40(3):599CrossRefGoogle Scholar
  26. 26.
    Zhang M, Li WB, Liu YX, Liang CJ, Liu LZ, Cui X, Gong JX, Gong SJ, Hu YY, Xian XH (2011) High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats. Neurochem Int 59(7):1019–1028CrossRefPubMedGoogle Scholar
  27. 27.
    Dalkara T, Moskowitz MA (1994) The complex role of nitric oxide in the pathophysiology of focal cerebral ischemia. Brain Pathol 4(1):49–57CrossRefPubMedGoogle Scholar
  28. 28.
    Kozniewska E, Roberts TP, Tsuura M, Mintorovitch J, Moseley ME, Kucharczyk J (1995) NG-nitro-L-arginine delays the development of brain injury during focal ischemia in rats. Stroke 26(2):282–288 (discussion 288–289)CrossRefPubMedGoogle Scholar
  29. 29.
    Lee KF, Chen JH, Teng CC, Shen CH, Hsieh MC, Lu CC, Lee KC, Lee LY, Chen WP, Chen CC, Huang WS, Kuo HC (2014) Protective effects of Hericium erinaceus mycelium and its isolated erinacine a against ischemia-injury-induced neuronal cell death via the inhibition of iNOS/p38 MAPK and nitrotyrosine. Int J Mol Sci 15(9):15073–15089CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Iadecola C, Anrather J (2011) The immunology of stroke: from mechanisms to translation. Nat Med 17(7):796–808CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Choe W, Kim S, Hwang TS, Lee SS (2003) Expression of inducible nitric oxide synthase in thyroid neoplasms: immunohistochemical and molecular analysis. Pathol Int 53(7):434–439CrossRefPubMedGoogle Scholar
  32. 32.
    Nanri K, Montécot C, Springhetti V, Seylaz J, Pinard E (1998) The selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole, reduces the delayed neuronal damage due to forebrain ischemia in rats. Stroke 29(6):1248–1253 (discussion 1253–1254)CrossRefPubMedGoogle Scholar
  33. 33.
    Gajkowska B, Viron A. Cholewiński M (1999) Immunocytochemical localization of endothelial nitric oxide synthase (e-NOS) and inducible nitric oxide synthase (i-NOS) in rat neurohypophysis after transient cerebral ischemia. Folia Neuropathol 37(1):10–19PubMedGoogle Scholar
  34. 34.
    Zhang M, Gong JX, Wang JL, Jiang MY, Li L, Hu YY, Qi J, Zhang LY, Zhao H, Cui X, Xian XH, Li WB (2017) p38 MAPK participates in the mediation of GLT-1 up-regulation during the induction of brain ischemic tolerance by cerebral ischemic preconditioning. Mol Neurobiol 54(1):58–71CrossRefPubMedGoogle Scholar
  35. 35.
    Lüth HJ, Holzer M, Gertz HJ, Arendt T (2000) Aberrant expression of nNOS in pyramidal neurons in Alzheimer’s disease is highly co-localized with p21ras and p16ink4a. Brain Res 852(1):45–55CrossRefPubMedGoogle Scholar
  36. 36.
    Katoh A, Kitazawa H, Itohara S, Nagao S (2000) Inhibition of nitric oxide synthesis and gene knockout of neuronal nitric oxide synthase impaired adaptation of mouse optokinetic response eye movements. Learn Mem 7(4):220–226CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Zeng WX, Han YL, Zhu GF, Huang LQ, Deng YY, Wang QS, Jiang WQ, Wen MY, Han QP, Xie D, Zeng HK (2017) Hypertonic saline attenuates expression of notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic bv-2 microglia. BMC Neurosci 18(1):32CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhou M, Wang CM, Yang WL, Wang P (2013) Microglial CD14 activated by iNOS contributes to neuroinflammation in cerebral ischemia. Brain Res 1506(2):105–114CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Ito N, Ruegg UT, Kudo A, Miyagoe-Suzuki Y, Takeda S (2013) Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy. Nat Med 19(1):101–106CrossRefPubMedGoogle Scholar
  40. 40.
    Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA (1994) Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265(5180):1883–1885CrossRefPubMedGoogle Scholar
  41. 41.
    Feng RF, Hu YY, Li WB, Liu HQ, Li QJ, Zhang M (2009) The role of no resulted from neuronal nitric oxide synthase in the metabotropic glutamate receptor2/3 mediated-brain ischemic tolerance. Zhongguo Ying Yong Sheng LI Xue Za Zhi 25(2):182–185PubMedGoogle Scholar
  42. 42.
    Ste-Marie L, Hazell AS, Bémeur C, Butterworth R, Montgomery J (2001) Immunohistochemical detection of inducible nitric oxide synthase, nitrotyrosine and manganese superoxide dismutase following hyperglycemic focal cerebral ischemia. Brain Res 918(1–2):10–19CrossRefPubMedGoogle Scholar
  43. 43.
    Yang C, Lai H, Zhan C, Xiao Y, Zheng W (2002) nNOS expression of hippocampal neurons in aged rats after brain ischemia/reperfusion and its role in DND development. Chin J Traumatol 5(4):232–236PubMedGoogle Scholar
  44. 44.
    Liu W, Li J, Sun X, Liu K, Zhang JH, Xu W, Tao H (2008) Repetitive hyperbaric oxygen exposures enhance sensitivity to convulsion by upregulation of eNOS and nNOS. Brain Res 1201(2):128–134CrossRefPubMedGoogle Scholar
  45. 45.
    Kim KH, Kim JI, Han JA, Choe MA, Ahn JH (2011) Upregulation of neuronal nitric oxide synthase in the periphery promotes pain hypersensitivity after peripheral nerve injury. Neuroscience 190:367–378CrossRefPubMedGoogle Scholar
  46. 46.
    Iadecola C, Zhang F, Casey R, Clark HB, Ross ME (1996) Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia. Stroke 27(8):1373–1380CrossRefPubMedGoogle Scholar
  47. 47.
    Bi XY, Wang TS, Zhang M, Liu QQ, Li WB, Zhang Y (2014) The up-regulation of p-p38 MAPK during the induction of brain ischemic tolerance induced by intermittent hypobaric hypoxia preconditioning in rats. Zhongguo Ying Yong Sheng Li Xue Za Zhi 30(2):97–100PubMedGoogle Scholar
  48. 48.
    Udayabanu M, Kumaran D, Nair RU, Srinivas P, Bhagat N, Aneja R, Anju K (2008) Nitric oxide associated with iNOS expression inhibits acetylcholinesterase activity and induces memory impairment during acute hypobaric hypoxia. Brain Res 1230(14):138–149CrossRefPubMedGoogle Scholar
  49. 49.
    Shi Q, Liu X, Wang N, Zheng X, Ran J, Liu Z, Fu J, Zheng J (2017) 1400W ameliorates acute hypobaric hypoxia/reoxygenation-induced cognitive deficits by suppressing the induction of inducible nitric oxide synthase in rat cerebral cortex microglia. Behav Brain Res 319:188–199CrossRefPubMedGoogle Scholar
  50. 50.
    Balez R, Ooi L (2015) Getting to no Alzheimer’s disease: neuroprotection versus neurotoxicity mediated by nitric oxide. Oxid Med Cell Longev 2016(4):1–8Google Scholar
  51. 51.
    Gupta SP, Kamal R, Mishra SK, Singh MK, Shukla R, Singh MP (2016) Association of polymorphism of neuronal nitric oxide synthase gene with risk to Parkinson’s disease. Mol Neurobiol 53(5):3309–3314CrossRefPubMedGoogle Scholar
  52. 52.
    Maiti P, Singh SB, Ilavazhagan G (2010) Nitric oxide system is involved in hypobaric hypoxia-induced oxidative stress in rat brain. Acta Histochem 112(3):222–232CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ya-Jie Huang
    • 1
  • Yu-Jia Yuan
    • 1
  • Yi-Xian Liu
    • 2
  • Meng-Yue Zhang
    • 1
  • Jing-Ge Zhang
    • 3
    Email author
  • Tian-Ci Wang
    • 1
  • Li-Nan Zhang
    • 3
  • Yu-Yan Hu
    • 3
  • Li Li
    • 4
  • Xiao-Hui Xian
    • 3
  • Jie Qi
    • 3
  • Min Zhang
    • 3
    • 5
    Email author
  1. 1.Undergraduate of Clinical MedicineHebei Medical UniversityShijiazhuangPeople’s Republic of China
  2. 2.Department of PhysiologyHebei Medical UniversityShijiazhuangPeople’s Republic of China
  3. 3.Department of PathophysiologyHebei Medical UniversityShijiazhuangPeople’s Republic of China
  4. 4.Department of Science and TechnologyThe Second Hospital of Hebei Medical UniversityShijiazhuangPeople’s Republic of China
  5. 5.Aging and Cognition Neuroscience Laboratory of Hebei ProvinceShijiazhuangPeople’s Republic of China

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