Cellular and Molecular Neurobiology

, Volume 31, Issue 2, pp 293–301 | Cite as

Activation of Different Neuronal Phenotypes in the Rat Brain Induced by Liver Ischemia–Reperfusion Injury: Dual Fos/Neuropeptide Immunohistochemistry

  • J. Bundzikova
  • Z. Pirnik
  • L. Lackovicova
  • B. Mravec
  • A. Kiss
Original Research


The aim of the present study was to reveal the effect of liver ischemia–reperfusion injury (LIRI) on the activity of selected neuronal phenotypes in rat brain by applying dual Fos-oxytocin (OXY), vasopressin (AVP), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), corticoliberine (CRH), and neuropeptide Y (NPY) immunohistochemistry. Two liver ischemia–reperfusion models were investigated: (i) single ligation of the hepatic artery (LIRIa) for 30 min and (ii) combined ligation of the portal triad (the common hepatic artery, portal vein, and common bile duct) (LIRIb) for 15 min. The animals were killed 90 min, 5 h, and 24 h after reperfusion. Intact and sham operated rats served as controls. As indicated by semiquantitative estimation, increases in the number of Fos-positive cells mainly occurred 90 min after both liver reperfusion injuries, including activation of AVP and OXY perikarya in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and TH, NPY, and PNMT perikarya in the catecholaminergic ventrolateral medullar A1/C1 area. Moreover, only PNMT perikarya located in the A1/C1 cell group exhibited increased Fos expression 5 h after LIRIb reperfusion. No or very low Fos expression was found 24 h after reperfusion in neuronal phenotypes studied. Our results show that both models of the LIRI activate, almost by the same effectiveness, a number of different neuronal phenotypes which stimulation may be associated with a complex of physiological responses induced by (1) surgery (NPY, TH, PNMT), (2) hemodynamic changes (AVP, OXY, TH, PNMT), (3) inflammation evoked by ischemia and subsequent reperfusion (TH), and (4) glucoprivation induced by fasting (NPY, PNMT, TH). All these events may contribute by different strength to the development of pathological alterations occurring during the liver ischemia–reperfusion injury.


Liver ischemia–reperfusion injury Fos Oxytocin Vasopressin Tyrosine Hydroxylase Phenylethanolamine N-methyltransferase Neuropeptide Y Dual immunohistochemistry Brain Rat 



The authors would like to thank Dr. Jens D. Mikkelsen and Dr. Greti Aguilera for the kind providing Fos, NPY, TH, OXY and AVP, CRH, PNMT antibodies, respectively. This study was supported by grant of the Ministry of Health of the Slovak Republic (MZ 2006/19-SAV-01) entitled “Stimulation of the vagus nerve as a new method for prevention of ischemia–reperfusion injury of transplanted organs”.


  1. Aoe T, Inaba H, Kon S, Imai M, Aono M, Mizuguchi T, Saito T, Nishino T (1997) Heat shock protein 70 messenger RNA reflects the severity of ischemia/hypoxia-reperfusion injury in the perfused rat liver. Crit Care Med 25:324–329PubMedCrossRefGoogle Scholar
  2. Bailey SM, Reinke LA (2000) Effect of low flow ischemia-reperfusion injury on liver function. Life Sci 66:1033–1044PubMedCrossRefGoogle Scholar
  3. Berthoud HR (2004) Anatomy and function of sensory hepatic nerves. Anat Rec A Discov Mol Cell Evol Biol 280:827–835PubMedCrossRefGoogle Scholar
  4. Berthoud HR, Neuhuber WL (2000) Functional and chemical anatomy of the afferent vagal system. Auton Neurosci 85:1–17PubMedCrossRefGoogle Scholar
  5. Bonaz B, Taché Y (1997) Corticotropin-releasing factor and systemic capsaicin-sensitive afferents are involved in abdominal surgery-induced Fos expression in the paraventricular nucleus of the hypothalamus. Brain Res 748:12–20PubMedCrossRefGoogle Scholar
  6. Bonnet MS, Pecchi E, Trouslard J, Jean A, Dallaporta M, Troadec JD (2009) Central nesfatin-1-expressing neurons are sensitive to peripheral inflammatory stimulus. J Neuroinflamm 6:27CrossRefGoogle Scholar
  7. Crockett ET, Spielman W, Dowlatshahi S, He J (2006) Sex differences in inflammatory cytokine production in hepatic ischemia-reperfusion. J Inflamm (Lond) 3:16CrossRefGoogle Scholar
  8. Duval H, Mbatchi SF, Grandadam S, Legendre C, Loyer P, Ribault C, Piquet-Pellorce C, Guguen-Guillouzo C, Boudjema K, Corlu A (2010) Reperfusion stress induced during intermittent selective clamping accelerates rat liver regeneration through JNK pathway. J Hepatol 52:560–569PubMedCrossRefGoogle Scholar
  9. Eum HA, Cha YN, Lee SM (2007) Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats. Biochem Biophys Res Commun 358:500–505PubMedCrossRefGoogle Scholar
  10. Frangogiannis NG (2007) Chemokines in ischemia and reperfusion. Thromb Haemost 97:738–747PubMedGoogle Scholar
  11. Girn HR, Ahilathirunayagam S, Mavor AI, Homer-Vanniasinkam S (2007) Reperfusion syndrome: cellular mechanisms of microvascular dysfunction and potential therapeutic strategies. Vasc Endovascular Surg 41:277–293PubMedCrossRefGoogle Scholar
  12. Gourcerol G, Gallas S, Mounien L, Leblanc I, Bizet P, Boutelet I, Leroi AM, Ducrotte P, Vaudry H, Jegou S (2007) Gastric electrical stimulation modulates hypothalamic corticotropin-releasing factor-producing neurons during post-operative ileus in rat. Neuroscience 148:775–781PubMedCrossRefGoogle Scholar
  13. Grindstaff RJ, Grindstaff RR, Cunningham JT (2000) Baroreceptor sensitivity of rat supraoptic vasopressin neurons involves noncholinergic neurons in the DBB. Am J Physiol Regul Integr Comp Physiol 279:R1934–R1943PubMedGoogle Scholar
  14. Gu GB, Ju G (1995) The parabrachio-subfornical organ projection in the rat. Brain Res Bull 38:41–47PubMedCrossRefGoogle Scholar
  15. Han F, Zhang YF, Li YQ (2003) Fos expression in tyrosine hydroxylase-containing neurons in rat brainstem after visceral noxious stimulation: an immunohistochemical study. World J Gastroenterol 9:1045–1050PubMedGoogle Scholar
  16. Hokfelt T, Johansson O, Goldstein M (1984) Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In: Bjorklund A, Hokfelt T (eds) Handbook of chemical neuroanatomy, vol 2. Elsevier, Amsterdam, pp 157–276Google Scholar
  17. Ionescu DA, Lugoji G, Radula D (1986) The nucleus of the solitary tract: a review of its anatomy and functions, with emphasis on its role in a putative central-control of brain-capillaries permeability. Neurol Psychiatr (Bucur) 24:69–85Google Scholar
  18. Jaeschke H, Bautista AP, Spolarics Z, Spitzer JJ (1992) Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats. J Leukoc Biol 52:377–382PubMedGoogle Scholar
  19. Kaur C, You Y, Singh J, Peng CM, Ling EA (2001) Expression of Fos immunoreactivity in some catecholaminergic brainstem neurons in rats following high-altitude exposure. J Neurosci Res 63:54–63PubMedCrossRefGoogle Scholar
  20. Kiss A, Aguilera G (1992) Participation of alpha 1-adrenergic receptors in the secretion of hypothalamic corticotropin-releasing hormone during stress. Neuroendocrinology 56:153–160PubMedCrossRefGoogle Scholar
  21. Kiss A, Søderman A, Bundzikova J, Pirnik Z, Mikkelsen JD (2006) Zolpidem, a selective GABAA receptor α1 subunit agonist, induces Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat. Brain Res Bull 71:200–207Google Scholar
  22. Kobashi M, Adachi A (1986) Projection of nucleus tractus solitarius units influenced by hepatoportal afferent signal to parabrachial nucleus. J Auton Nerv Syst 16:153–158PubMedCrossRefGoogle Scholar
  23. Krukoff TL, MacTavish D, Harris KH, Jhamandas JH (1995) Changes in blood volume and pressure induce c-fos expression in brainstem neurons that project to the paraventricular nucleus of the hypothalamus. Brain Res Mol Brain Res 34:99–108PubMedCrossRefGoogle Scholar
  24. Lacroix S, Rivest S (1997) Functional circuitry in the brain of immune-challenged rats: partial involvement of prostaglandins. J Comp Neurol 387:307–324PubMedCrossRefGoogle Scholar
  25. Li AJ, Ritter S (2004) Glukoprivation increases expression of neuropeptide Y mRNA in hindbrain neurons that innervate hypothalamus. Eur J Neurosci 19:2147–2154PubMedCrossRefGoogle Scholar
  26. Li SQ, Liang LJ, Huang JF, Li Z (2003) Hepatocyte apoptosis induced by hepatic ischemia-reperfusion injury in cirrhotic rats. Hepatobiliary Pancreat Dis Int 2(1):102–105PubMedGoogle Scholar
  27. Luo X, Kiss A, Makara G, Lolait SJ, Aguilera G (1994) Stress-specific regulation of corticotropin releasing hormone receptor expression in the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Neuroendocrinol 6:689–696PubMedCrossRefGoogle Scholar
  28. Martins PN, Neuhaus P (2007) Surgical anatomy of the liver, hepatic vasculature and bile ducts in the rat. Liver Int 27:384–392PubMedCrossRefGoogle Scholar
  29. Mikkelsen JD, Vrang N, Mrosovsky N (1998) Expression of Fos in the circadian system following nonphotic stimulation. Brain Res Bull 47:367–376PubMedCrossRefGoogle Scholar
  30. Miyatake Y, Ikeda H, Michimataa R, Koizumia S, Ishizua A, Nishimura N, Yoshiki T (2004) Differential modulation of gene expression among rat tissues with warm ischemia. Exp Mol Pathol 77:222–230PubMedCrossRefGoogle Scholar
  31. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Academic Press, New York, USAGoogle Scholar
  32. Pirnik Z, Kiss A (2005) Fos expression variances in mouse hypothalamus upon physical and osmotic stimuli: Co-staining with vasopressin, oxytocin, and tyrosine hydroxylase. Brain Res Bull 65:423–431PubMedCrossRefGoogle Scholar
  33. Pirnik Z, Mravec B, Kiss A (2004a) Fos protein expression in mouse hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei upon osmotic stimulus: colocalization with vasopressin, oxytocin, and tyrosine hydroxylase. Neurochem Int 45:597–607PubMedCrossRefGoogle Scholar
  34. Pirnik Z, Mravec B, Kubovcakova L, Mikkelsen JD, Kiss A (2004b) Hypertonic saline and immobilization induce Fos expression in mouse brain catecholaminergic cell groups: colocalization with tyrosine hydroxylase and neuropeptide Y. Ann N Y Acad Sci 1018:398–404PubMedCrossRefGoogle Scholar
  35. Pirnik Z, Bundzikova J, Francisty T, Cibulova E, Lackovicova L, Mravec B, Kiss A (2009) Effect of ischemia-reperfusion of liver on the activity of neurons in the rat brain. Cell Mol Neurobiol 29:951–960PubMedCrossRefGoogle Scholar
  36. Rhodes CH, Morrell JI, Pfaff DW (1981) Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin. J Comp Neurol 198:45–64PubMedCrossRefGoogle Scholar
  37. Ritter S, Llewellyn-Smith I, Dinh TT (1998) Subgroups of hindbrain catecholamine neurons are selectively activated by 2-deoxy-d-glucose induced metabolic challenge. Brain Res 805:41–54PubMedCrossRefGoogle Scholar
  38. Romani F, Vertemati M, Frangi M, Aseni P, Monti R, Codeghini A, Belli L (1988) Effect of superoxide dismutase on liver ischemia-reperfusion injury in the rat: a biochemical monitoring. Eur Surg Res 20:335–340PubMedCrossRefGoogle Scholar
  39. Shioda S, Nakai Y (1996) Direct projections form catecholaminergic neurons in the caudal ventrolateral medulla to vasopressin-containing neurons in the supraoptic nucleus: a triple-labeling electron microscope study in the rat. Neurosci Lett 221:45–48PubMedCrossRefGoogle Scholar
  40. Shioda S, Shimoda Y, Nakai Y (1992) Ultrastructural studies of medullary synaptic inputs to vasopressin-immunoreactive neurons in the supraoptic nucleus of the rat hypothalamus. Neurosci Lett 148:155–158PubMedCrossRefGoogle Scholar
  41. Spiegel HU, Bahde R (2006) Experimental models of temporary normothermic liver ischemia. J Invest Surg 19:113–123PubMedCrossRefGoogle Scholar
  42. Stanley S, Pinto S, Segal J, Pérez CA, Viale A, DeFalco J, Cai X, Heisler LK, Friedman JM (2010) Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically. Proc Natl Acad Sci USA 107:7024–7029PubMedCrossRefGoogle Scholar
  43. Steiner PE, Martinez JB (1961) Effects on the rat liver of bile duct, portal vein and hepatic artery ligations. Am J Pathol 39:257–289PubMedGoogle Scholar
  44. Sved AF, Mancini DL, Graham JC, Schreihofer AM, Hoffman GE (1994) PNMT-containing neurons of the C1 cell group express c-fos in response to changes in baroreceptor input. Am J Physiol 266:R361–R367PubMedGoogle Scholar
  45. Swanson LW, Kuypers HG (1980) The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194:555–570PubMedCrossRefGoogle Scholar
  46. Swanson LW, Sawchenko PE, Wiegand SJ, Price JL (1980) Separate neurons in the paraventricular nucleus project to the median eminence and to the medulla or spinal cord. Brain Res 198:190–195PubMedCrossRefGoogle Scholar
  47. Teoh NC, Farrell GC (2003) Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol 18:891–902PubMedCrossRefGoogle Scholar
  48. Uyama N, Geerts A, Reynaert H (2004) Neural connections between the hypothalamus and the liver. Anat Rec A Discov Mol Cell Evol Biol 280:808–820PubMedCrossRefGoogle Scholar
  49. Walsh KB, Toledo AH, Rivera-Chavez FA, Lopez-Neblina F, Toledo-Pereyra LH (2009) Inflammatory mediators of liver ischemia–reperfusion injury. Exp Clin Transpl 7:78–93Google Scholar
  50. Wanner GA, Ertel W, Müller P, Höfer Y, Leiderer R, Menger MD, Messme K (1996) Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation. Shock 5:34–40PubMedCrossRefGoogle Scholar
  51. Xiao JS, Cai FG, Niu Y, Zhang Y, Xu XL, Ye QF (2005) Preconditioning effects on expression of proto-oncogenes c-fos and c-jun after hepatic ischemia/reperfusion in rats. Hepatobiliary Pancreat Dis Int 4:197–202PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • J. Bundzikova
    • 1
  • Z. Pirnik
    • 1
    • 2
  • L. Lackovicova
    • 1
  • B. Mravec
    • 1
    • 3
  • A. Kiss
    • 1
  1. 1.Laboratory of Functional NeuromorphologyInstitute of Experimental Endocrinology, Slovak Academy of SciencesBratislavaSlovak Republic
  2. 2.Department of Pharmacy and Pharmaceutical TechnologyUniversity of Veterinary Medicine and PharmacyKosiceSlovak Republic
  3. 3.Institute of Pathological PhysiologyFaculty of Medicine, Comenius UniversityBratislavaSlovak Republic

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