Background Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats. Methods On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH2O (pH = 8.5) was injected into the CM at 10 μg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test. Results The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (P<0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (P = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test. Conclusion By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.
References
[1]
American Academy of Pediatrics (2004) Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 114: 297–316. doi: 10.1542/peds.114.1.297
[2]
Johnson L, Bhutani VK, Karp k, Sivieri EM, Shapiro SM (2009) Clinical report from the pilot USA Kernicterus Registry (1992 to 2004)Root causes of kernicterus. J Perinatol 29 Suppl 1S25–45. doi: 10.1038/jp.2008.211
[3]
Manning D, Todd P, Maxwell M, Jane Platt M (2007) Prospective surveillance study of severe hyperbilirubinaemia in the newborn in the United Kingdom and Ireland. Arch Dis Child Fetal Neonatal Ed 92: F342–F346. doi: 10.1136/adc.2006.105361
[4]
Sgro M, Campbell DM, Kandasamy S, Shah V (2012) Incidence of chronic bilirubin encephalopathy in Canada, 2007–2008. Pediatrics 130(4): e886–90. doi: 10.1542/peds.2012-0253
[5]
Liu KZ, He HY, Hua ZY (2012) Clinical analysis of acute bilirubin encephalopathy in 227 neonates. J Clin Pediatr (in Chinese) 30(9): 840–844.
[6]
Gunn CH (1938) Hereditary acholuric jaundice in a new mutant strain of rats. J Hered 29: 137–139.
[7]
Blanc WA, Johnson L (1959) Studies on kernicterus: relationship with sulfonamide intoxication, report on kernicterus in rats with glucuronyl transferase deficiency and review of pathogenesis. J Neuropathol Exp Neurol 18: 165–189. doi: 10.1097/00005072-195901000-00011
[8]
Rose AL, Wisniewski H (1979) Acute bilirubin encephalopathy induced with sulfadimethoxine in Gunn rats. J Neuropathol Exp Neurol 38: 152–165. doi: 10.1097/00005072-197903000-00006
[9]
Shapiro SM (1988) Acute brainstem auditory evoked potential abnormalities in jaundiced Gunn rats given sulfonamide. Pediatr Res 23: 306–310. doi: 10.1203/00006450-198803000-00015
[10]
Rice AC, Shapiro SM (2008) A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus). Pediatr Res 64(3): 265–269. doi: 10.1203/pdr.0b013e31817d9be0
[11]
Mejia GB, Sanz CR, Avila MM, Peraza AV, Guzmán DC, et al. (2008) Experimental hemolysis model to study bilirubin encephalopathy in rat brain. J Neurosci Metho 168: 35–41. doi: 10.1016/j.jneumeth.2007.09.003
[12]
Mottino AD, Pellegrino JM, Guibert EE, Roma MG, Rodriguez Garay EA (1988) Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes. Int J Biochem 20: 1113–1116. doi: 10.1016/0020-711x(88)90256-x
[13]
Li XD, Xia SQ, Lv Y, He P, Han J, et al. (2004) Conjugation metabolism of acetaminophen and bilirubin in extrahepatic tissues of rats. Life Sci 74: 1307–1315. doi: 10.1016/j.lfs.2003.08.011
[14]
Nguyen N, Bonzo JA, Chen S, Chouinard S, Kelner MJ, et al. (2008) Disruption of the Ugt1 locus in mice resembles human Crigler-Najjar type I disease. J Biol Chem 283: 7901–7911. doi: 10.1074/jbc.m709244200
[15]
Fujiwara R, Nguyen N, Chen S, Tukey RH (2010) Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus. Proc Natl Acad Sci U S A 107(11): 5024–5029. doi: 10.1073/pnas.0913290107
Rainov NG, Kramm CM (2001) Vector delivery methods and targeting strategies for gene therapy of brain tumors. Curr Gene Ther 1: 367–383. doi: 10.2174/1566523013348445
[18]
Mahat MY, Fakrudeen Ali Ahamed N, Chandrasekaran S, Rajagopal S, Narayanan S, et al. (2012) An improved method of transcutaneous cisterna magna puncture for cerebrospinal fluid sampling in rats. J Neurosci Methods 211: 272–279. doi: 10.1016/j.jneumeth.2012.09.013
[19]
Hemsley KM, King B, Hopwood JJ (2007) Injection of recombinant human sulfamidase into the CSF via the cerebellomedullary cistern in MPS IIIA mice. Mol Genet Metab 90: 313–328. doi: 10.1016/j.ymgme.2006.10.005
[20]
Proescholdt MG, Hutto B, Brady LS, Herkenham M (2000) Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat. Neuroscience 95: 577–592. doi: 10.1016/s0306-4522(99)00417-0
[21]
Wennberg RP, Hance AJ (1986) Experimental bilirubin encephalopathy: importance of total bilirubin, protein binding, and blood-brain barrier. Pediatr Res 20: 789–792. doi: 10.1203/00006450-198608000-00018
[22]
Chen S, Owens GC, Crossin KL, Edelman DB (2007) Serotonin stimulates mitochondrial transport in hippocampal neurons. Mol Cell Neurosci 36: 472–483. doi: 10.1016/j.mcn.2007.08.004
[23]
Goldberg JE, Sherwood SW, Clayberger C (1999) A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry. J Immunol Methods 224: 1–9. doi: 10.1016/s0022-1759(98)00038-6
[24]
Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11: 47–60. doi: 10.1016/0165-0270(84)90007-4
[25]
Yang Y, Cao J, Xiong W, Zhang J, Zhou Q, et al. (2003) Both stress experience and age determine the impairment or enhancement effect of stress on spatial memory retrieval. J Endocrinol 178: 45–54. doi: 10.1677/joe.0.1780045
[26]
Johnson LH, Bhutani VK, Brown AK (2002) System-based approach to management of neonatal jaundice and prevention of kernicterus. J Pediatr 140: 396–403. doi: 10.1067/mpd.2002.123098
[27]
Springer SC, Annibale DJ (2012) Kernicterus. Medscape Reference. Available: http://emedicine.medscape.com/article/97？5276-overview. Updated: Apr 2012.
[28]
Ahdab-Barmada M, Moossy J (1984) The neuropathology of kernicterus in the premature neonate: diagnostic problems. J Neuropathol Exp Neurol 43: 45–56. doi: 10.1097/00005072-198401000-00004
[29]
Rossato JI, Bevilaqua LR, Myskiw JC, Medina JH, Izquierdo I, et al. (2007) On the role of hippocampal protein synthesis in the consolidation and reconsolidation of object recognition memory. Learn Mem 14: 36–46. doi: 10.1101/lm.422607
[30]
Jarrard LE (1993) On the role of the hippocampus in learning and memory in the rat. Behav Neural Bio 60: 9–26. doi: 10.1016/0163-1047(93)90664-4
[31]
Watchko JF (2006) Kernicterus and the molecular mechanisms of bilirubin-induced CNS injury in newborns. Neuromolecular Med 8: 513–29. doi: 10.1385/nmm:8:4:513
[32]
Brites D (2012) The evolving landscape of neurotoxicity by unconjugated bilirubin: role of glial cells and inflammation. Front Pharmacol 3: 88. doi: 10.3389/fphar.2012.00088
[33]
Sunico CR, González-Forero D, Domínguez G, García-Verdugo JM, Moreno-López B (2010) Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinase-dependent mechanism preceded by myosin light chain phosphorylation. J Neurosci 30: 973–984. doi: 10.1523/jneurosci.3911-09.2010
Rodrigues CM, Solá S, Brites D (2002) Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology 35: 1186–1195. doi: 10.1053/jhep.2002.32967
[36]
Hank？ E, Hansen TW, Almaas R, Lindstad J, Rootwelt T (2004) Bilirubin induces apoptosis and necrosis in human NT2-N neurons. Pediatr Res 57: 179–184. doi: 10.1203/01.pdr.0000148711.11519.a5
[37]
Watchko JF (2005) Bilirubin induced apoptosis in vitro: insights for kernicterus. Pediatr Res 57: 177–178. doi: 10.1203/01.pdr.0000150727.50378.ea
[38]
Cardoso FL, Kittel á, Veszelka S, Palmela I, Tóth A, et al. (2012) Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells. PLoS One 7: e35919. doi: 10.1371/journal.pone.0035919
[39]
Zhang TJ, Hang J, Wen DX, Hang YN, Sieber FE (2006) Hippocampus bcl-2 and bax expression and neuronal apoptosis after moderate hypothermic cardiopulmonary bypass in rats. Anesth Analg 102: 1018–1025. doi: 10.1213/01.ane.0000199221.96250.8c
[40]
Northington FJ, Graham EM, Martin LJ (2005) Apoptosis in perinatal hypoxic-ischemic brain injury: how important is it and should it be inhibited. Brain Res Brain Res Rev 50: 244–257. doi: 10.1016/j.brainresrev.2005.07.003
