Recombinant hirudin treatment modulates aquaporin-4 and aquaporin-9 expression after intracerebral hemorrhage in vivo

  • Zhe Sun
  • Zhenhuan Zhao
  • Shiguang Zhao
  • Yi Sheng
  • Zonghang Zhao
  • Cheng Gao
  • Jingwen Li
  • Xiangzhen Liu


Edema formation has been linked to thrombin toxicity induced by blood clot at the acute stage of intracerebral hemorrhage. Thrombin induces cell toxicity in neuron, microglia and astrocyte. Aquaporin (AQP) 4 and 9 are proteins expressed on astrocyte in rat brain and involved in the brain water accumulation in brain edema. Recombinant hirudin (r-Hirudin) is a direct inhibitor of thrombin that can block the toxicitic effect of thrombin. In this study, we demonstrated that autologous whole blood infusion in caudate nucleus up-regulates the expression of AQP4 and AQP9 mRNAs and proteins. AQP4 and AQP9 mRNAs expression peaked at about 6 h after blood infusion. The AQP4 protein peaked at about 48 h while AQP9 at about 24 h after blood infusion. Thrombin induced up-regulation of AQP4 and AQP9 were inhibited by r-Hirudin administration and significantly decreased the expression of both AQPs. We further investigated the relationship between edema formation and expression of AQP4 and AQP9. The data presented here may be helpful in optimizing r-Hirudin as an anti-thrombin drug in the treatment of edema at the acute stage of ICH.


Recombinant hirudin Thrombin Aquaporin Autologous whole blood 



This study was supported by grants D200530 from the Province Nature Science Foundation.


  1. 1.
    Lee KR, Colon GP, Betz AL et al (1996) Edema from intracerebral hemorrhage: the role of thrombin. J Neurosurg 84(1):91–96PubMedGoogle Scholar
  2. 2.
    Xi G, Wagner KR, Keep RF et al (1998) Role of blood clot formation on early edema development following experimental intracerebral hemorrhage. Stroke 29(12):2580–2586PubMedGoogle Scholar
  3. 3.
    Loret C, Sensenbrenner M, Labourdette G (1989) Differential phenotypic expression induced in cultured rat astroblasts by acidic fibroblast growth factor, epidermal growth factor, and thrombin. J Biol Chem 264(14):8319–8327PubMedGoogle Scholar
  4. 4.
    Gurwitz D, Cunningham DD (1988) Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci USA 85(10):3440–3444. doi: 10.1073/pnas.85.10.3440 PubMedCrossRefGoogle Scholar
  5. 5.
    Lee KR, Betz AL, Keep RF et al (1995) Intracerebral infusion of thrombin as a cause of brain edema. J Neurosurg 83(6):1045–1050PubMedCrossRefGoogle Scholar
  6. 6.
    Lee KR, Betz AL, Kim S et al (1996) The role of the coagulation cascade in brain edema formation after intracerebral hemorrhage. Acta Neurochir (Wien) 138(4):396–401. doi: 10.1007/BF01420301 CrossRefGoogle Scholar
  7. 7.
    Venero JL, Vizuete ML, Machado A et al (2001) Aquaporins in the central nervous system. Prog Neurobiol 63(3):321–336. doi: 10.1016/S0301-0082(00)00035-6 PubMedCrossRefGoogle Scholar
  8. 8.
    Badaut J, Petit JM, Brunet JF et al (2004) Distribution of aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells. Neuroscience 128(1):27–38. doi: 10.1016/j.neuroscience.2004.05.042 PubMedCrossRefGoogle Scholar
  9. 9.
    Jung JS, Bhat RV, Preston GM et al (1994) Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA 91(26):13052–13056. doi: 10.1073/pnas.91.26.13052 PubMedCrossRefGoogle Scholar
  10. 10.
    Venero JL, Vizuete ML, Ilundain AA et al (1999) Detailed localization of aquaporin-4 messenger RNA in the CNS: preferential expression in periventricular organs. Neuroscience 94(1):239–250. doi: 10.1016/S0306-4522(99)00182-7 PubMedCrossRefGoogle Scholar
  11. 11.
    Hiroaki Y, Tani K, Kamegawa A et al (2006) Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol 355(4):628–639. doi: 10.1016/j.jmb.2005.10.081 PubMedCrossRefGoogle Scholar
  12. 12.
    Nielsen S, Nagelhus EA, Amiry-Moghaddam M et al (1997) Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17(1):171–180PubMedGoogle Scholar
  13. 13.
    de Castro Ribeiro M, Hirt L, Bogousslavsky J et al (2006) Time course of aquaporin expression after transient focal cerebral ischemia in mice. J Neurosci Res 83(7):1231–1240. doi: 10.1002/jnr.20819 CrossRefGoogle Scholar
  14. 14.
    Ishibashi K, Kuwahara M, Gu Y et al (1998) Cloning and functional expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem Biophys Res Commun 244(1):268–274. doi: 10.1006/bbrc.1998.8252 PubMedCrossRefGoogle Scholar
  15. 15.
    Tsukaguchi H, Shayakul C, Berger UV et al (1998) Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem 273(38):24737–24743. doi: 10.1074/jbc.273.38.24737 PubMedCrossRefGoogle Scholar
  16. 16.
    Amiry-Moghaddam M, Lindland H, Zelenin S et al (2005) Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane. FASEB J 19(11):1459–1467. doi: 10.1096/fj.04-3515com PubMedCrossRefGoogle Scholar
  17. 17.
    Hamada R, Matsuoka H (2000) Antithrombin therapy for intracerebral hemorrhage. Stroke 31(3):791–795Google Scholar
  18. 18.
    Markwardt F (1994) The development of hirudin as an antithrombotic drug. Thromb Res 74(1):1–23. doi: 10.1016/0049-3848(94)90032-9 PubMedCrossRefGoogle Scholar
  19. 19.
    Koster A, Kuppe H, Hetzer R et al (1998) Emergent cardiopulmonary bypass in five patients with heparin-induced thrombocytopenia type II employing recombinant hirudin. Anesthesiology 89(3):777–780. doi: 10.1097/00000542-199809000-00029 PubMedCrossRefGoogle Scholar
  20. 20.
    Lubenow N, Eichler P, Lietz T et al (2004) Lepirudin for prophylaxis of thrombosis in patients with acute isolated heparin-induced thrombocytopenia: an analysis of 3 prospective studies. Blood 104(10):3072–3077. doi: 10.1182/blood-2004-02-0621 PubMedCrossRefGoogle Scholar
  21. 21.
    Mehta SR, Eikelboom JW, Rupprecht HJ et al (2002) Efficacy of hirudin in reducing cardiovascular events in patients with acute coronary syndrome undergoing early percutaneous coronary intervention. Eur Heart J 23(2):117–123. doi: 10.1053/euhj.2001.2731 PubMedCrossRefGoogle Scholar
  22. 22.
    Choi TS, Khan AI, Greilich PE et al (2006) Modified plasma-based ecarin clotting time assay for monitoring of recombinant hirudin during cardiac surgery. Am J Clin Pathol 125(2):290–295. doi: 10.1309/0TDG-8BEC-LWCB-XCDK PubMedGoogle Scholar
  23. 23.
    Steinbrich W, Gross-Fengels W, Krestin GP et al (1990) Intracranial hemorrhages in the magnetic resonance tomogram: studies on sensitivity, on the development of hematomas and on the determination of the cause of the hemorrhage. Rofo 152(5):534–543PubMedGoogle Scholar
  24. 24.
    Lin TN, He YY, Wu G et al (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24(1):117–121PubMedGoogle Scholar
  25. 25.
    Yang GY, Betz AL, Chenevert TL et al (1994) Experimental intracerebral hemorrhage: relationship between brain edema, blood flow, and blood-brain barrier permeability in rats. J Neurosurg 81(1):93–102PubMedGoogle Scholar
  26. 26.
    Xi GH, Keep RF, Hua Y et al (1999) Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning. Stroke 30(6):1247–1255PubMedGoogle Scholar
  27. 27.
    Arima H, Yamamoto N, Sobue K et al (2003) Hyperosmolar mannitol stimulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes. J Biol Chem 278(45):44525–44534. doi: 10.1074/jbc.M304368200 PubMedCrossRefGoogle Scholar
  28. 28.
    Koeppen AH, Dickson AC, McEvoy JA (1995) The cellular reactions to experimental intracerebral hemorrhage. J Neurol Sci 34(suppl 1):102–112. doi: 10.1016/0022-510X(95)00215-N CrossRefGoogle Scholar
  29. 29.
    Karabiyikoglu M, Hua Y, Keep RF et al (2004) Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab 24(2):159–166. doi: 10.1097/01.WCB.0000100062.36077.84 PubMedCrossRefGoogle Scholar
  30. 30.
    Donovan FM, Pike CJ, Cotman CW et al (1997) Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. Neuroscience 17(14):5316–5326PubMedGoogle Scholar
  31. 31.
    Ke C, Poon WS, Ng HK et al (2001) Heterogeneous responses of aquaporin-4 in oedema formation in a replicated severe traumatic brain injury model in rats. Neurosci Lett 301(1):21–24. doi: 10.1016/S0304-3940(01)01589-0 PubMedCrossRefGoogle Scholar
  32. 32.
    Kiening KL, van Landeghem FK, Schreiber S et al (2002) Decreased hemispheric aquaporin-4 is linked to evolving brain edema following controlled cortical impact injury in rats. Neurosci Lett 324(2):105–108. doi: 10.1016/S0304-3940(02)00180-5 PubMedCrossRefGoogle Scholar
  33. 33.
    Badaut J, Hirt L, Granziera C et al (2001) Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia. J Cereb Blood Flow Metab 21(5):477–482. doi: 10.1097/00004647-200105000-00001 PubMedCrossRefGoogle Scholar
  34. 34.
    Hua Y, Xi G, Keep RF et al (2000) Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg 92(6):1016–1022PubMedGoogle Scholar
  35. 35.
    Xi GH, Hua Y, Richard FK et al (2001) Systemic complement depletion diminishes perihematomal brain edema in rats. Stroke 32(1):162–167. doi: 10.1161/hs1201.099820 PubMedCrossRefGoogle Scholar
  36. 36.
    Manley GT, Fujimura M, Ma T et al (2000) Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med 6(2):159–163. doi: 10.1038/72256 PubMedCrossRefGoogle Scholar
  37. 37.
    Rash JE, Yasumura T, Hudson CS et al (1998) Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc Natl Acad Sci USA 95(20):11981–11986. doi: 10.1073/pnas.95.20.11981 PubMedCrossRefGoogle Scholar
  38. 38.
    Papadopoulos MC, Manley GT, Krishna S et al (2004) Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J 18(11):1291–1293PubMedGoogle Scholar
  39. 39.
    Badaut J, Regli L (2004) Distribution and possible roles of aquaporin 9 in the brain. Neuroscience 129(4):971–981. doi: 10.1016/j.neuroscience.2004.06.035 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Zhe Sun
    • 1
  • Zhenhuan Zhao
    • 1
  • Shiguang Zhao
    • 1
  • Yi Sheng
    • 1
  • Zonghang Zhao
    • 2
  • Cheng Gao
    • 1
  • Jingwen Li
    • 1
  • Xiangzhen Liu
    • 1
  1. 1.Harbin Medical UniversityHarbinChina
  2. 2.University of CalgaryCalgaryCanada

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