Clinical and Experimental Pediatrics

대한소아청소년과학회 (The Korean Pediatric Society)
권호 표지
DOI QR코드

DOI QR Code

신생 백서의 저산소 허혈 뇌손상에서 Transforming Growth Factor-β1 투여에 따른 Nitric Oxide Synthase 이성체와 N-methyl-D-aspartate 수용체 아단위의 발현

Expression of nitric oxide synthase isoforms and N-methyl-D-aspartate receptor subunits according to transforming growth factor-β1 administration after hypoxic-ischemic brain injury in neonatal rats

  • 고혜영 (구미강동병원 소아청소년과) ;
  • 서억수 (동국대학교 의과대학 안과학교실) ;
  • 김우택 (대구가톨릭대학교 의과대학 소아과학교실)
  • Go, Hye Young (Department of Pediatrics, Gumi-Gangdong Hospital) ;
  • Seo, Eok Su (Dongguk University College of Medicine) ;
  • Kim, Woo Taek (Department of Pediatrics, School of Medicine, Catholic University of Daegu)
  • 투고 : 2008.12.24
  • 심사 : 2009.03.13
  • 발행 : 2009.05.15
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

목 적 : $TGF-{\beta}1$는 흥분독성을 억제시키고 질소 산화물 생성 억제를 통한 신경세포 보호 효과가 있다고 알려져 있지만 주산기저산소 허혈 뇌손상에서 그 기전은 아직도 확실히 밝혀져 있지 않고 있다. 따라서 이번 연구에서는 신생 백서의 저산소 허혈 뇌손상에서 산화질소로 인한 신경독성 및 글루탐산염에 의한 흥분독성과 $TGF-{\beta}1$의 관계를 보고자 하였다. 방 법 : 생체외 실험으로 재태 기간 19일된 태아 백서의 대뇌피질 세포를 배양하여 1% O2 배양기에서 저산소 상태로 뇌세포손상을 유도하여 저산소군(Hypoxia), 저산소 손상 30분 전 $TGF-{\beta}1$ (1, 5, 10 ng/mL) 투여군(H+$TGF-{\beta}1$)으로 나누어 정상 산소군 (Control)과 비교하였다. 생체 내 실험은 생후 7일된 백서의 좌측 총 경동맥을 결찰한 후 저산소 (7.5% O2) 상태로 2시간 노출시켜서, 저산소 허혈 뇌손상을 유발하였다. 아무런 처치도 하지 않은 정상 대조군(Control), 경동맥 노출 후 봉합 시술만 시행한 정상 Sham 수술군(Sham-OP), 손상 30분 전 생리식염수를 주입 후 경동맥 결찰과 저산소 노출을 시행한 저산소 허혈 대조군(HI+ Vehicle), 손상 30분 전 $TGF-{\beta}1$을 대뇌로 투여하고 경동맥 결찰과 저산소 노출을 시행한 저산소 허혈 $TGF-{\beta}1$ 투여군(HI+$TGF-{\beta}1$)으로 나누어 비교분석하였다. 흥분독성과의 관련을 알아보기 위하여 NMDA 수용체 아단위를 이용하였고, 질소산화물과의 관련을 알아보기 위해 iNOS, eNOS 및 nNOS를 이용하여 western blotting과 실시간 중합효소연쇄반응을 하였다. 결 과 : 생체 외 실험에서 iNOS의 발현은 정상 산소군과 저산소군 간에 차이가 없었으며, $TGF-{\beta}1$ 투여군에서는 발현이 증가하였으며 이는 농도와는 상관성이 없었다. eNOS, nNOS의 발현은 1 ng/mL의 $TGF-{\beta}1$ 투여군에서 저산소군보다 감소하였다. 생체 내 실험에서는 iNOS와 iNOS mRNA의 발현은 $TGF-{\beta}1$ 투여한 후 저산소 대조군보다 증가하였다. eNOS와 nNOS 발현은 정상 대조군 보다 저산소 대조군에서 감소하였고, eNOS의 발현은 $TGF-{\beta}1$ 투여군에서 증가하였지만 nNOS의 발현은 증가하지 않아 통계적 유의성이 없었다. eNOS mRNA와 nNOS mRNA의 발현은 iNOS와 반대로 $TGF-{\beta}1$ 투여군에서 저산소 대조군보다 감소하였다. NMDA 수용체 아단위 mRNA의 발현은 정상 대조군과 Sham 수술군에 비해 저산소 대조군에서 모두 감소하였으나 $TGF-{\beta}1$ 투여군에서 NR2C를 제외한 나머지 아단위의 발현은 저산소 대조군보다 증가하였다. 결 론 : 신생백서의 저산소 허혈 뇌손상에서 $TGF-{\beta}1$ 치료군에서 저산소로 인하여 감소된 NMDA 수용체 아단위의 발현을 증가시켜 흥분독성 기전과 관련성을 보이며, 증가된 iNOS 발현을 감소시키고 감소된 eNOS 발현을 증가시키는 질소 산화물 중재를 통한 뇌 보호 작용에 연관이 있을 것으로 생각된다.

Purpose : Transforming growth factor (TGF)-${\beta}1$ reportedly increases neuronal survival by inhibiting the induction of inducible nitric oxide synthase (NOS) in astrocytes and protecting neurons after excitotoxic injury. However, the neuroprotective mechanism of $TGF-{\beta}1$ on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether $TGF-{\beta}1$ has neuroprotective effects via a NO-mediated mechanism and N-methyl-D-aspartate (NMDA) receptor modulation on perinatal HI brain injury. Methods : Cortical cells were cultured using 19-day-pregnant Sprague-Dawley (SD) rats treated with $TGF-{\beta}1$ (1, 5, or 10 ng/mL) and incubated in a 1% O2 incubator for hypoxia. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 h of hypoxic exposure (7.5% $O_2$). $TGF-{\beta}1$ (0.5 ng/kg) was administered intracerebrally to the rats 30 min before HI brain injury. The expressions of NOS and NMDA receptors were measured. Results : In the in vitro model, the expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS) increased in the hypoxic group and decreased in the 1 ng/mL $TGF-{\beta}1-treated$ group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the $TGF-{\beta}1$-treated group. The expressions of eNOS and nNOS were reversed compared with the expression of iNOS. The expressions of all NMDA receptor subunits decreased in hypoxia group and increased in the $TGF-{\beta}1$-treated group except NR2C. Conclusion : The administration of $TGF-{\beta}1$ could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.

키워드

참고문헌

  1. Berger R, Garnier Y. Pathophysiology of perinatal brain damage. Brain Res Brain Res Rev 1999;30:107-34 https://doi.org/10.1016/S0165-0173(99)00009-0
  2. Towfighi J, Mauger D, Vannucci R.C, Vannucci SJ. Influence of age on the cerebral lesions in an immature rat model of cerebral hypoxia　ischemia: a light microscopic study. Brain Res Dev Brain Res 1997;100:149-60 https://doi.org/10.1016/S0165-3806(97)00036-9
  3. Tan S, Zhou F, Nielsen VG, Wang Z, Gladson CL, Parks DA. Sustained hypoxia-ischemia results in reactive nitrogen and oxygen species production and injury in the premature fetal rabbit brain. J Neuropathol Exp Neurol 1998;57:544-53 https://doi.org/10.1097/00005072-199806000-00002
  4. 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
  5. Peruche B, Krieglstein J. Mechanisms of drug actions against neuronal damage caused by ischemia--an overview. Prog Neuropsychopharmacol Biol Psychiatry 1993;17:21-70 https://doi.org/10.1016/0278-5846(93)90032-N
  6. Cull-Candy SG, Leszkiewicz DN. Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004;255:re16
  7. Moncada S, Palmer RM, Higgs E.A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43: 109-42
  8. Iadecola C, Pelligrino DA, Moskowitz MA, Lassen NA. Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab 1994;14:175-92 https://doi.org/10.1038/jcbfm.1994.25
  9. Stagliano NE, Zhao W, Prado R, Dewanjee MK, Ginsberg MD, Dietrich WD. The effect of nitric oxide synthase inhibition on acute platelet accumulation and hemodynamic depression in a rat model of thromboembolic stroke. J Cereb Blood Flow Metab 1997;17:1182-90
  10. Massagu$\acute{e}$ J. TGFbeta signaling: receptors, transducers, and Mad proteins. Cell 1996;85:l947-50
  11. Lorez H, Keller F, Ruess G, Otten U. Nerve growth factor increases in adult rat brain after hypoxic injury. Neurosci Lett 1989;98:339-44 https://doi.org/10.1016/0304-3940(89)90425-4
  12. Williams C, Guan J, Miller O, Beilharz E, McNeill H, Sirimanne E, Gluckman P. The role of the growth factors IGF- 1 and TGF beta 1 after hypoxic-ischemic brain injury. Ann N Y Acad Sci 1995;765:306-7 https://doi.org/10.1111/j.1749-6632.1995.tb16592.x
  13. Wiessner C, Gehrmann J, Lindholm D, T$\ddot{o}$pper R, Kreutzberg GW, Hossmann KA. Expression of transforming growth factor-beta 1 and interleukin-1 beta mRNA in rat brain following transient forebrain ischemia. Acta Neuropathol 1993; 86:439-46 https://doi.org/10.1007/BF00228578
  14. Gross CE, Bednar MM, Howard DB, Sporn MB. Transforming growth factor-beta 1 reduces infarct size after experimental cerebral ischemia in a rabbit model. Stroke 1993;24: 558-62
  15. Vivien D, Bernaudin M, Buisson A, Divoux D, MacKenzie ET, Nouvelot A. Evidence of type I and type II transforming growth factor-beta receptors in central nervous tissues: changes induced by focal cerebral ischemia. J Neurochem 1998;70:2296-304 https://doi.org/10.1046/j.1471-4159.1998.70062296.x
  16. Flanders KC, Ren RF, Lippa CF. Transforming growth factor-betas in neurodegenerative disease. Prog Neurobiol 1998; 54:71-85 https://doi.org/10.1016/S0301-0082(97)00066-X
  17. Chung HM, Choi EJ, Seo ES, Kim WT. The neuroprotective effect of transforming growth factor-beta1 via anti-apoptosis on hypoxic-ischemic brain injury in neonatal rats. Korean J Perinatol 2008;19:42-53
  18. Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 1997;71:143-55 https://doi.org/10.1016/S0165-0270(96)00136-7
  19. 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
  20. Vannucci RC. Hypoxic-ischemic encephalopathy. Am J Perinatol 2000;17:113-20 https://doi.org/10.1055/s-2000-9293
  21. Romijn HJ, Hofman MA, Gramsbergen A. At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby? Early Hum Dev 1991;26:61-7 https://doi.org/10.1016/0378-3782(91)90044-4
  22. Naito S, Ueda T. Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles. J Biol Chem 1983;258:696-9
  23. Dienel G. Hertz L. Astrocytic contributions to bioenergetics of cerebral ischemia. Glia 2005;50:362-88 https://doi.org/10.1002/glia.20157
  24. Prehn JH, Bindokas VP, Marcuccilli CJ, Krajewski S, Reed JC, Miller RJ. Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons. Proc Natl Acad Sci USA 1994;91:12599-603 https://doi.org/10.1073/pnas.91.26.12599
  25. Buisson A, Nicole O, Docagne F, Sartelet H, Mackenzie ET, Vivien D. Up-regulation of a serine protease inhibitor in astrocytes mediates the neuroprotective activity of transforming growth factor beta1. FASEB J 1998;12:1683-91
  26. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. NMDA- dependent superoxide production and neurotoxicity. Nature 1993;364:535-7 https://doi.org/10.1038/364535a0
  27. Hollmann M, Heinemann S. Cloned glutamate receptors. Annu Re. Neurosci 1994;17:31-108 https://doi.org/10.1146/annurev.ne.17.030194.000335
  28. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361: 31-9 https://doi.org/10.1038/361031a0
  29. Malenka R, Nicoll RA. NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms. Trends Neurosci 1993;16:521-7 https://doi.org/10.1016/0166-2236(93)90197-T
  30. Nicoll RA, Malenka RC. Expression mechanisms underlying NMDA receptor-dependent long-term potentiation. Ann N Y Acad Sci 1999;868:515-25 https://doi.org/10.1111/j.1749-6632.1999.tb11320.x
  31. Tsien JZ, Huerta PT, Tonegawa S. The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 1996;87:1327-38 https://doi.org/10.1016/S0092-8674(00)81827-9
  32. Choi DW. Excitotoxic cell death. J Neurobiol 1992;23:1261-76 https://doi.org/10.1002/neu.480230915
  33. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 1997;20:132-9 https://doi.org/10.1016/S0166-2236(96)10074-6
  34. Samdani AF, Dawson TM, Dawson VL. Nitric oxide synthase in models of focal ischemia. Stroke 1997;28:1283-8
  35. Parathath S, Parathath S, Tsirka S. Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice. J Cell Sci 2006; 119:339-49 https://doi.org/10.1242/jcs.02734
  36. Zollner S, Aberle S, Harvey SE, Polokoff MA, Rubanyi GM. Changes of endothelial nitric oxide synthase level and activity during endothelial cell proliferation. Endothelium 2000; 7:169-84 https://doi.org/10.3109/10623320009165315
  37. Wong D, Dorovini-Zis K, Vincent S. Cytokines. nitric oxide and CGMP modulate the permeability of an in vitro model of the human blood　brain barrier. Exp Neurol 2004;190:446-55 https://doi.org/10.1016/j.expneurol.2004.08.008
  38. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, et al. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab 1996;16:981-7
  39. Dimmeler S, Dernbach E, Zeiher A. Phosphorylation of the endothelial nitric oxide synthase at Ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett 2000; 477:258-62 https://doi.org/10.1016/S0014-5793(00)01657-4
  40. Dawson V, Kizushi V, Huang P, Snyder S, Dawson T. Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. J Neurosci 1996;16:2479-87

피인용 문헌

  1. Effects of Dizocilpine (MK-801) via Up-modulation of N-methyl-D-aspartate (NMDA) Receptors on Hypoxic-Ischemic Brain Injury in Neonatal Rats vol.25, pp.3, 2009, https://doi.org/10.14734/kjp.2014.25.3.166