Experimental Brain Research

, Volume 212, Issue 1, pp 109–117 | Cite as

Effects of hyperbaric oxygen on the expression of claudins after cerebral ischemia–reperfusion in rats

  • Hong Zhao
  • Qianru Zhang
  • Yixue Xue
  • Xuexin Chen
  • Randy S. Haun
Research Article


The malfunction of tight junctions (TJs) between endothelial cells in the blood brain barrier (BBB) is the pathophysiological basis for cerebral ischemia–reperfusion (IR) injury. Claudins, major molecular elements of the TJs, play a key role in the paracellular permeability of the BBB. Although several studies have demonstrated the impact of hyperbaric oxygenation (HBO) on boosting oxygen supply and reducing infarct size, its effect and underlying mechanism on the integrity of the BBB is unknown. To study the function of HBO on claudins and the permeability of the BBB, we replicated the animal model of local cerebral IR. Using Evans blue dye, permeability of the BBB was examined. Transmission electron microscopy (TEM), immunohistochemistry, western blot, and gelatin zymography were used to detect the integrity of the BBB, the expression of claudin-1 and claudin-5, and the activity of matrix metalloproteinases (MMPs) in brain microvessel endothelium. Our data indicate that compared with the sham-operated group, IR increased permeability of the BBB to Evans blue dye (P < 0.01), peaking at 4 h. The BBB ultrastructure was disrupted and the expression of claudin-5 and claudin-1 decreased (P < 0.01) in the 4 and 72 h IR group, respectively. Increased claudin-5 and claudin-1 expression and decreased permeability of the BBB were observed in the HBO + IR group (P < 0.01) via the suppression of MMP-2 and MMP-9, respectively. Our study provides direct evidence that HBO decreases the permeability of the BBB by reducing the enzymatic activity of MMPs and augmenting the expression of claudins at different stages in cerebral IR injury.


Blood brain barrier Ischemia–reperfusion Hyperbaric oxygen Claudin 



Adherens junction


Blood brain barrier


Common carotid artery


Evans blue


External carotid artery


Hyperbaric oxygenation


Horseradish peroxidase


Internal carotid artery




Middle cerebral artery occlusion


Matrix metalloproteinase


Reverse transcription polymerase chain reaction


Streptoavidin–biotin complex


Tight junction



This work was supported by the Natural Science Foundation of China, No. 30570650, 30670723, and 30700861; Natural Science Foundation of Liaoning Province, No. 20052102; Foundation of Liaoning Educational Committee, No. 2005L456; and Liaoning Province Doctoral Startup Fund No. 20101149.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Allan SM, Parker LC, Collins B, Davies R, Luheshi GN, Rothwell NJ (2000) Cortical cell death induced by IL-1 is mediated via actions in the hypothalamus of the rat. Proc Natl Acad Sci USA 97:5580–5585PubMedCrossRefGoogle Scholar
  2. Andras IE, Pu H, Tian J, Deli MA, Nath A, Hennig B, Toborek M (2005) Signaling mechanisms of HIV-1 Tat-induced alterations of claudin-5 expression in brain endothelial cells. J Cereb Blood Flow Metab 25:1159–1170PubMedCrossRefGoogle Scholar
  3. Atochin DN, Fisher D, Demchenko IT, Thom SR (2000) Neutrophil sequestration and the effect of hyperbaric oxygen in a rat model of temporary middle cerebral artery occlusion. Undersea Hyperb Med 27:185–190PubMedGoogle Scholar
  4. Ballabh P, Braun A, Nedergaard M (2004) The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 16:1–13PubMedCrossRefGoogle Scholar
  5. Campbell SJ, Carare-Nnadi RO, Losey PH, Anthony DC (2007) Loss of the atypical inflammatory response in juvenile and aged rats. Neuropathol Appl Neurobiol 33:108–120PubMedCrossRefGoogle Scholar
  6. Cucullo L, McAllister MS, Kight K, Krizanac-Bengez L, Marroni M, Mayberg MR, Stanness KA, Janigro D (2002) A new dynamic in vitro model for the multidimensional study of astrocyte-endothelial cell interactions at the blood-brain barrier. Brain Res 951:243–254PubMedCrossRefGoogle Scholar
  7. Davies B, Miles DW, Happerfield LC, Naylor MS, Bobrow LG, Rubens RD, Balkwill FR (1993) Activity of type IV collagenases in benign and malignant breast disease. Br J Cancer 67:1126–1131PubMedCrossRefGoogle Scholar
  8. Durukan A, Tatlisumak T (2007) Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav 87:179–197PubMedCrossRefGoogle Scholar
  9. Freedman FB, Johnson JA (1969) Equilibrium and kinetic properties of the Evans blue-albumin system. Am J Physiol 216:675–681PubMedGoogle Scholar
  10. Gao D, Zhang X, Jiang X, Peng Y, Huang W, Cheng G, Song L (2006) Resveratrol reduces the elevated level of MMP-9 induced by cerebral ischemia-reperfusion in mice. Life Sci 78:2564–2570PubMedCrossRefGoogle Scholar
  11. Gasche Y, Copin JC, Sugawara T, Fujimura M, Chan PH (2001) Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia. J Cereb Blood Flow Metab 21:1393–1400PubMedCrossRefGoogle Scholar
  12. Gunther A, Kuppers-Tiedt L, Schneider PM, Kunert I, Berrouschot J, Schneider D, Rossner S (2005) Reduced infarct volume and differential effects on glial cell activation after hyperbaric oxygen treatment in rat permanent focal cerebral ischaemia. Eur J Neurosci 21:3189–3194PubMedCrossRefGoogle Scholar
  13. Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173–185PubMedCrossRefGoogle Scholar
  14. Kleiner DE, Stetler-Stevenson WG (1994) Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem 218:325–329PubMedCrossRefGoogle Scholar
  15. Kuyvenhoven JP, Ringers J, Verspaget HW, Lamers CB, van Hoek B (2003) Serum matrix metalloproteinase MMP-2 and MMP-9 in the late phase of ischemia and reperfusion injury in human orthotopic liver transplantation. Transplant Proc 35:2967–2969PubMedCrossRefGoogle Scholar
  16. Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, Wolburg H (2000a) Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100:323–331PubMedCrossRefGoogle Scholar
  17. Liebner S, Kniesel U, Kalbacher H, Wolburg H (2000b) Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. Eur J Cell Biol 79:707–717PubMedCrossRefGoogle Scholar
  18. Lohmann C, Krischke M, Wegener J, Galla HJ (2004) Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways. Brain Res 995:184–196PubMedCrossRefGoogle Scholar
  19. Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91PubMedCrossRefGoogle Scholar
  20. Lou M, Eschenfelder CC, Herdegen T, Brecht S, Deuschl G (2004) Therapeutic window for use of hyperbaric oxygenation in focal transient ischemia in rats. Stroke 35:578–583PubMedCrossRefGoogle Scholar
  21. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109–142PubMedGoogle Scholar
  22. Mrsic-Pelcic J, Pelcic G, Vitezic D, Antoncic I, Filipovic T, Simonic A, Zupan G (2004) Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+, K+ -ATPase activities in global cerebral ischemia-exposed rats. Neurochem Int 44:585–594PubMedCrossRefGoogle Scholar
  23. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660PubMedCrossRefGoogle Scholar
  24. Nordal RA, Wong CS (2005) Molecular targets in radiation-induced blood-brain barrier disruption. Int J Radiat Oncol Biol Phys 62:279–287PubMedCrossRefGoogle Scholar
  25. Ostrowski RP, Jadhav V, Chen W, Zhang JH (2010) Reduced matrix metalloproteinase-9 activity and cell death after global ischemia in the brain preconditioned with hyperbaric oxygen. Acta Neurochir Suppl 106:47–49PubMedCrossRefGoogle Scholar
  26. Persidsky Y, Heilman D, Haorah J, Zelivyanskaya M, Persidsky R, Weber GA, Shimokawa H, Kaibuchi K, Ikezu T (2006) Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE). Blood 107:4770–4780PubMedCrossRefGoogle Scholar
  27. Planas AM, Sole S, Justicia C (2001) Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia. Neurobiol Dis 8:834–846PubMedCrossRefGoogle Scholar
  28. Poli S, Veltkamp R (2009) Oxygen therapy in acute ischemic stroke—experimental efficacy and molecular mechanisms. Curr Mol Med 9:227–241PubMedCrossRefGoogle Scholar
  29. Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC (1998) Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke 29:1020–1030PubMedCrossRefGoogle Scholar
  30. Shyamaladevi N, Jayakumar AR, Sujatha R, Paul V, Subramanian EH (2002) Evidence that nitric oxide production increases gamma-amino butyric acid permeability of blood-brain barrier. Brain Res Bull 57:231–236PubMedCrossRefGoogle Scholar
  31. Soini Y (2005) Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology 46:551–560PubMedCrossRefGoogle Scholar
  32. Stevenson BR, Keon BH (1998) The tight junction: morphology to molecules. Annu Rev Cell Dev Biol 14:89–109PubMedCrossRefGoogle Scholar
  33. Tsukita S, Furuse M (2000) Pores in the wall: claudins constitute tight junction strands containing aqueous pores. J Cell Biol 149:13–16PubMedCrossRefGoogle Scholar
  34. Ueno M (2007) Molecular anatomy of the brain endothelial barrier: an overview of the distributional features. Curr Med Chem 14:1199–1206PubMedCrossRefGoogle Scholar
  35. Veltkamp R, Siebing DA, Sun L, Heiland S, Bieber K, Marti HH, Nagel S, Schwab S, Schwaninger M (2005) Hyperbaric oxygen reduces blood-brain barrier damage and edema after transient focal cerebral ischemia. Stroke 36:1679–1683PubMedCrossRefGoogle Scholar
  36. Veltkamp R, Sun L, Herrmann O, Wolferts G, Hagmann S, Siebing DA, Marti HH, Veltkamp C, Schwaninger M (2006) Oxygen therapy in permanent brain ischemia: potential and limitations. Brain Res 1107:185–191PubMedCrossRefGoogle Scholar
  37. Vlodavsky E, Palzur E, Feinsod M, Soustiel JF (2005) Evaluation of the apoptosis-related proteins of the BCL-2 family in the traumatic penumbra area of the rat model of cerebral contusion, treated by hyperbaric oxygen therapy: a quantitative immunohistochemical study. Acta Neuropathol 110:120–126PubMedCrossRefGoogle Scholar
  38. Waisman D, Shupak A, Weisz G, Melamed Y (1998) Hyperbaric oxygen therapy in the pediatric patient: the experience of the Israel Naval Medical Institute. Pediatrics 102:E53PubMedCrossRefGoogle Scholar
  39. Wang CX, Yang Y, Yang T, Shuaib A (2001) A focal embolic model of cerebral ischemia in rats: introduction and evaluation. Brain Res Brain Res Protoc 7:115–120PubMedCrossRefGoogle Scholar
  40. Welgus HG, Campbell EJ, Cury JD, Eisen AZ, Senior RM, Wilhelm SM, Goldberg GI (1990) Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development. J Clin Invest 86:1496–1502PubMedCrossRefGoogle Scholar
  41. Woessner JF Jr (1994) The family of matrix metalloproteinases. Ann N Y Acad Sci 732:11–21PubMedCrossRefGoogle Scholar
  42. Yamamoto M, Ramirez SH, Sato S, Kiyota T, Cerny RL, Kaibuchi K, Persidsky Y, Ikezu T (2008) Phosphorylation of claudin-5 and occludin by rho kinase in brain endothelial cells. Am J Pathol 172:521–533PubMedCrossRefGoogle Scholar
  43. Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA (2007) Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J Cereb Blood Flow Metab 27:697–709PubMedCrossRefGoogle Scholar
  44. Yin D, Zhou C, Kusaka I, Calvert JW, Parent AD, Nanda A, Zhang JH (2003) Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model. J Cereb Blood Flow Metab 23:855–864PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hong Zhao
    • 1
  • Qianru Zhang
    • 1
  • Yixue Xue
    • 2
  • Xuexin Chen
    • 3
  • Randy S. Haun
    • 4
  1. 1.Department of Experimental Center of Functional Subjects, College of Basic MedicineChina Medical UniversityShenyangChina
  2. 2.Department of Neurobiology, College of Basic MedicineChina Medical UniversityShenyangChina
  3. 3.Department of Hyperbaric OxygenThe 1st Affiliated Hospital of China Medical UniversityShenyangChina
  4. 4.Department of Pathology, College of MedicineUniversity of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations