Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
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Neuroprotective effects of geneticin (G418) via apoptosis in perinatal hypoxic-ischemic brain injury

주산기 저산소성 허혈성 뇌손상에서 항고사를 통한 geneticin (G418)의 신경보호 효과

  • Ju, Mi (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Lee, Hyun Ju (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Lee, Sun Ju (Department of Pediatrics, School of Medicine, DongGuk University) ;
  • Seo, Eo Su (Department of Ophthalmology, School of Medicine, DongGuk University) ;
  • Park, Hye Jin (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Lee, Kye Yang (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Lee, Gyeong Hoon (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Choi, Eun Jin (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Kim, Jin Kyung (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Lee, Jong Won (Department of Biochemistry, School of Medicine, Catholic University of Daegu) ;
  • Chung, Hai Lee (Department of Pediatrics, School of Medicine, Catholic University of Daegu) ;
  • Kim, Woo Taek (Department of Pediatrics, School of Medicine, Catholic University of Daegu)
  • 주미 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 이현주 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 이선주 (동국대학교 의과대학 소아과학교실) ;
  • 서억수 (동국대학교 의과대학 안과학교실) ;
  • 박혜진 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 이계향 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 이경훈 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 최은진 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 김진경 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 이종원 (대구가톨릭대학교 의과대학 생화학교실) ;
  • 정혜리 (대구가톨릭대학교 의과대학 소아과학교실) ;
  • 김우택 (대구가톨릭대학교 의과대학 소아과학교실)
  • Received : 2007.12.04
  • Accepted : 2008.01.09
  • Published : 2008.02.15
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Abstract

Purpose : Some antibiotics were known to exert neuroprotective effects in the animal model of hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. A recent study reported that geneticin (G418), an aminoglycoside antibiotic, increased survival of human breast cancer cells by suppressing apoptosis. We investigated the neuroprotective effects of systemically administrated geneticin via anti-apoptosis following the H-I brain injury Methods : Seven-day-old Sprague-Dawley rat pups were subjected to unilateral (left) common carotid artery occlusion followed by 2.5 hours of hypoxic exposure and the cortical cell culture of rat brain was done under a hypoxic incubator. Apoptosis was measured in the injured hemispheres 7 days after H-I insult and in the injured cells from hypoxic chamber using morphologic analysis by Terminal dUTP Nick-end Labeling(TUNEL) assay and immunohistochemistry for caspase-3, and cytologic analysis by western blot and real time PCR for bax, bcl-2, and caspase-3. Results : The gross appearance and hematoxylin and eosin stain revealed increased brain volume in the geneticin-treated animal model of perinatal H-I brain injury. The TUNEL assay revealed decreased apoptotic cells after administration of geneticin in the cell culture model of anoxia. Immunohistochemistry showed decreased caspase-3 expression in geneticin-treated cortical cell culture. Western blot and real-time PCR showed decreased caspase-3 expression and decreased ratio of Bax/Bcl-2 expression in geneticin-treated animal model. Conclusion : Geneticin appears to exert a neuroprotective effect against perinatal H-I brain injury at least via anti-apoptosis. However, more experiments are needed in order to demonstrate the usefulness of geneticin as a preventive and rescue treatment for H-I brain injuries of neonatal brain.

목 적 : 몇몇 항생제가 저산소성 허혈성 뇌 손상에서 뇌 보호 효과를 가진 것으로 밝혀졌지만 아직까지 그 기전에 대해 잘 알려지지 않고 있다. 최근 아미노글루코사이드 계열의 항생제인 G418(geneticin)이 항고사에 의한 암세포 생존을 증가 시키는 것으로 알려졌으며 본 연구는 G418이 주산기 저산소성 허혈성 뇌손상에서 세포 고사를 억제함으로서 뇌 보호 효과를 나타내는지를 알아보고자 하였다. 방 법 : 임신 18일된 백서의 대뇌피질 신경세포를 배양하여 정상산소 상태군와 저산소 상태군으로 나누고, 두 군을 각각 대조군과 G418 $10{\mu}g/mL$으로 처리한 군으로 나누어서 TUNEL 분석과 caspase 3에 대한 면역조직생화학검사를 하였고, 생후 7일된 신생 백서의 좌측 총경동맥을 결찰 후 절단하고 저산소 상태를 유도하여 G418을 저산소 상태 유도 전 30분과 유도 후 30분에 복강 내로 $0.1{\mu}g/kg$ 투여하고 7일 후에 희생시켜 H&E 염색과 caspase-3에 대해 Western blot과 real-time PCR를 하였다. 결 과 : TENEL 분석상 정상산소 상태군과 저산소 상태군 모두에서 대조군보다 G418 투여군에서 통계학적으로 유의하게 고사세포가 감소되었다(P<0.01). Caspase-3에 대한 면역조직화학검사에서 저산소 손상 전에 G418을 투여한 군에서 손상 후에 투여한 군보다 Caspase-3 발현이 적게 나타났다. 저산소 손상 7일 후에 얻은 신생 백서 뇌의 육안적 관찰과 H & E 염색에서 저산소 상태군의 뇌 용적이 정상산소 상태군의 경우보다 감소된 것을 알 수 있었고, 저산소 손상 전에 G418을 투여한 군에서 손상 후에 투여한 군보다 뇌 조직의 손상이 더 적은 것을 볼 수 있었다. Bax/Bcl-2, caspase-3에 대해 Western blot과 real-time PCR 한 경우에 G418 투여한 군에서 Bax/Bcl-2 비율과 caspase-3 발현이 감소되었다. 결 론 : 주산기 저산소성 허혈성 뇌 손상에서 G418는 뇌 조직의 고사를 억제함으로서 뇌보호 효과를 나타내었다.

Keywords

Acknowledgement

Supported by : 대구가톨릭대학교 의과학연구소

References

  1. Vannucci RC. Hypoxic-ischemic encephalopathy. Am J Perinatol 2000;17:113-20 https://doi.org/10.1055/s-2000-9293
  2. Vannucci RC, Perlman JM. Interventions for perinatal hypoxic-ischemic encephalopathy. Pediatrics 1997;100:1004-14 https://doi.org/10.1542/peds.100.6.1004
  3. Vannucci RC, Connor JR, Mauger DT, Palmer C, Smith MB, Towfighi J, et al. Rat model of perinatal hypoxic-ischemic brain damage. J Neurosci Res 1999;55:158-63 https://doi.org/10.1002/(SICI)1097-4547(19990115)55:2<158::AID-JNR3>3.0.CO;2-1
  4. Weitzdoerfer R, Pollak A, Lubec B. Perinatal asphyxia in the rat has lifelong effects on morphology, cognitive functions, and behavior. Semin Perinatol 2004;28:249-56 https://doi.org/10.1053/j.semperi.2004.08.001
  5. Calvert JW, Zhang JH. Pathophysiology of an hypoxic-ischemic insult during the perinatal period. Neurol Res 2005;27:246-60 https://doi.org/10.1179/016164105X25216
  6. Williams CE, Gunn A, Gluckman PD. Time course of intracellular edema and epileptiform activity following prenatal cerebral ischemia in sheep. Stroke 1991;22:516-21 https://doi.org/10.1161/01.STR.22.4.516
  7. Vannucci RC. Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediatr Res 1990;27:317-26 https://doi.org/10.1203/00006450-199004000-00001
  8. Han BH, Xu D, Choi J, Han Y, Xanthoudakis S, Roy S, et al. Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem 2002;277:30128-36 https://doi.org/10.1074/jbc.M202931200
  9. Yin W, Cao G, Johnnides MJ, Signore AP, Luo Y, Hickey RW, et al. TAT-mediated delivery of Bcl-xL protein is neuroprotective against neonatal hypoxic-ischemic brain injury via inhibition of caspases and AIF. Neurobiol Dis 2006;21:358-71 https://doi.org/10.1016/j.nbd.2005.07.015
  10. Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J. Tetracycline Inhibit Microglial Activation and Are Neuroprotective in Global Brain Ischemia. Proc Natl Acad Sci USA 1998;22:15769-74
  11. Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 1999;96:13496-500 https://doi.org/10.1073/pnas.96.23.13496
  12. Tikka T, Usenius T, Tenhunen M, Keinanen R, Koistinaho J. Tetracycline Derivatives and Ceftriaxone, A Cephalosporin Antibiotic, Protect Neurons Against Apoptosis Induced by Ionizing Radiation. J Neurochem 2001;78:1409-14 https://doi.org/10.1046/j.1471-4159.2001.00543.x
  13. Arvin KL, Han BH, Du Y, Lin SZ, Raul SM, Holtzman DM. Minocycline Markedly Protects the Neonatal Brain Against Hypoxic-Ischemia Injury. Ann Neurol 2002;52:54-61 https://doi.org/10.1002/ana.10242
  14. Jantzie LL, Cheung PY, Todd KG. Doxycycline reduces cleaved caspase-3 and microglial activation in an animal model of neonatal hypoxia-ischemia. J Cereb Blood Flow Metab 2005;25:314-24 https://doi.org/10.1038/sj.jcbfm.9600025
  15. Lee TS, Park SH, Lee JW, Lim SH. The effect of Geneticin on the survival of a human breast cancer cells under hypoxic condition. J Korean Breast Cancer Soc 2002;5:279-83 https://doi.org/10.4048/jkbcs.2002.5.4.279
  16. Levine S. Anoxic-ischemic encephalopathy in rats. Am J Pathol 1960;6:1-17
  17. Rice JE 3rd, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981;9:131-41 https://doi.org/10.1002/ana.410090206
  18. Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J Neurosci Meth 1997;71:143-55 https://doi.org/10.1016/S0165-0270(96)00136-7
  19. Loebenberg D, Counells M, Waitz JA. Antibiotic G-418, a New Micromonospora-Produced Aminoglycoside with Activity Against Protozoa and Helminths. Antimicrob Agents Chemother 1975;7:811-15 https://doi.org/10.1128/AAC.7.6.811
  20. Eustice DC, Wilhelm JM. Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis. Antimicrob Agents Chemother 1984;26:53-60 https://doi.org/10.1128/AAC.26.1.53
  21. Coggan JS, Kovacs I, Thompson SH. The aminiglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells. Neurosci Lett 1994;11:247-50 https://doi.org/10.1016/0304-3940(79)90002-8
  22. Volpe JJ. Perinatal brain injury: from pathogenesis to neuroprotection. Ment Retard Dev Disabil Res Rev 2001;7:56-64 https://doi.org/10.1002/1098-2779(200102)7:1<56::AID-MRDD1008>3.0.CO;2-A
  23. Johnston BM, Mallard EC, Willians CE, Gluckman PD. Insulin-like growth factor-1 is a potent neuronal rescue agent after hypoxic-ischemic injury in fetal lambs. J Clin Invest 1996;97:300-8 https://doi.org/10.1172/JCI118416
  24. Northington FJ, Ferriero DM, Graham EM, Traystman RJ, Martin LJ. Early Neurodegeneration after Hypoxia-Ischemia in Neonatal Rat Is Necrosis while Delayed Neuronal Death Is Apoptosis. Neurobiol Dis 2001;8:207-19 https://doi.org/10.1006/nbdi.2000.0371
  25. Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 1993;14:133-51
  26. Banasiak KJ, Xia Y, Haddad GG. Mechanisms underlying hypoxia-induced neuronal apoptosis. Prog Neurobiol 2000;62:215-49 https://doi.org/10.1016/S0301-0082(00)00011-3
  27. Hill IE, MacManus JP, Rasquinha I, Tuor UI. DNA fragmentaion indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat. Brain Res 1995;676:398-403 https://doi.org/10.1016/0006-8993(95)00145-G
  28. Nakajima W, Ishida A, Lange MS, Gabrielson KL, Wilson MA, Martin LJ, et al. Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat. J Neurosci 2000;20:7994-8004 https://doi.org/10.1523/JNEUROSCI.20-21-07994.2000
  29. White A. Systemic review of therapy after hypoxic-ischaemic brain injury in the perinatal period. Semin Neonatol 2000;5:33-40
  30. McDonald JW, Silverstein FS, Johnston MV. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Rev 1990;15:41-70 https://doi.org/10.1016/0165-0173(90)90011-C
  31. Ishida A, Trescher WH, Lange MS, Johnston MV. Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole pretects against cerebral hypoxic-ischemic injury in neonatal rat. Brain Dev 2001;23:349-54 https://doi.org/10.1016/S0387-7604(01)00237-6
  32. Thoresen M, Bagenholm R, Loberg EM, Apricena F, Kjellmer I. Post-hypoxic cooling of neonatal rats provides protection against brain injury. Arch Dis Child Fetal Neonatal Ed 1996;74:F3-F9 https://doi.org/10.1136/fn.74.1.F3
  33. Yasuoka N, Nakajima W, Ishida A, Takada G. Neuroprotection of Edaravone on hypoxic-ischemia brain injury in neonatal rats. Brain Res 2004;19:129-39
  34. Chen Y, Ginis I, Hallenbeck JM. The protective effect of ceramide in immature rat brain hypoxia-ischemia involves up-regulation of bcl-2 and reduction of TUNEL-positive cells. J Cereb Blood Flow Metab 2001;21:34-40 https://doi.org/10.1097/00004647-200101000-00005
  35. Turkyilmaz C, Turkyilmaz Z, Atalay Y, Soylemezoglu F, Celasun B. Magnesium pre-treatment reduces neuronal apoptosis in newborn rats in hypoxia-ischemia. Brain Res 2002;15:133-7
  36. Vannucci RC, Vannucci SJ. Perinatal hypoxic-ischemic brain damage: evolution of an animal model. Dev Neurosci 2005;27:81-6 https://doi.org/10.1159/000085978
  37. Clark WM, Calcagno FA, Gabler WL, Smith JR, Coull BM. Reduction of Central Nervous System Reperfusion Injury in Rabbits Using Doxycycline treatment. Stroke 1994;25:1411-5 https://doi.org/10.1161/01.STR.25.7.1411