臺灣博碩士論文加值系統

新生兒缺血窒息腦傷(neonatal hypoxic-ischemic brain injury)是指新生兒在出生前後因窒息缺氧引起的腦部損傷。目前為止，臨床上對於這種傷害並無有效的治療方式。「前置處理」(preconditioning)指的是在致命性的傷害發生之前，事先給予生物體一個非致命性的刺激，進而促使生物體產生對抗致命性傷害的保護機制而減少所受的傷害。了解「前置處理」的形成機制，可以提供臨床上可能預防及治療新生兒缺血窒息腦傷的研究方向。
將出生後七天的幼鼠進行單側頸動脈結紮並且合併8 %低氧氣濃度，會在同側腦部形成缺氧腦傷。為了解缺血前置處理是否有time- dependent的關係，我們建立缺血前置處理模式(ischemic preconditioning model，IP)，此模式是在傷害前事先在新生幼鼠右側頸動脈以血管夾夾住2小時，然後分別在放開血管夾後之2小時(IP-2h group)、6小時(IP-6h group)及22小時(IP-22h group)再讓幼鼠接受缺血窒息腦傷，在大白鼠35天及40天大時，分別利用行為測試及病理分析進行評估。
我們發現與未接受IP之大白鼠(No-IP group)比較，經過缺血前置處理之三組IP組別在學習和記憶的行為及病理分析腦傷嚴重度的評估中，皆顯示能形成明顯的腦部保護。而此三組IP組別的行為測試結果則顯示IP-6h的行為表現較其他兩組差，且由TUNEL stain同樣觀察到在三組IP中IP-6h有較多TUNEL positive細胞。
我們進一步發現與No-IP group之大白鼠比較，IP-22h group在缺血窒息傷害發生後24小時顯著地減少反應性氧自由基(reactive oxygen species，ROS)的表現量，並且可大量減少外在(extrinsic)及內在路徑(intrinsic pathway)中細胞凋亡之指標分子(pro-apoptotic markers)的活化情形，包括：caspase-8、caspase-3、caspase-9及poly (ADP-ribose) polymerase (PARP)。進一步往上游觀察到缺血前置處理可明顯地減少粒線體pro-apoptotic markers釋放到細胞質的現象，包括：cytochrome c、Smac及apoptosis inducing factor (AIF)之釋放。此外，缺血前置處理也會促使抗細胞凋亡指標分子(anti-apoptotic markers)之表現，包括Bcl-2及cellular inhibitor of apoptosis-1 (cIAP-1)；值得注意的是是cIAP-1在前置處理後會在大腦皮質(cortex)及海馬迴(hippocampus)有明顯地增加表現的情形，而X-chromosome linked IAP (XIAP)則沒有明顯的變化。
本實驗首次在新生鼠腦中建立了缺血前置處理模式，並且證實由缺血前置處理所達成的腦部保護的情形不僅包括減少細胞凋亡機制之活化的發生(caspase-dependent和caspase-independent pathways，及intrinsic 和extrinsic pathways)；同時也可經由增加抗細胞凋亡(anti-apoptosis)相關蛋白的表現來達到神經保護作用，我們特別發現cIAP-1在缺血前置處理腦部保護機制中的角色值得深入探討。

Neonatal hypoxic-ischemic (HI) brain injury is a major cause of neonatal mortality and long-term disability. Currently, there are still no effective therapies against neonatal HI brain injury. A sublethal stress, such as a brief episode of ischemia, before a lethal injury may reduce neuronal death against subsequent lethal injury; a phenomenon called “preconditioning”. Elucidating the underlying mechanisms of preconditioning may provide potential neuroprotective therapy for neonatal HI brain injury.
Permanent ligation of unilateral carotid artery followed by systemic hypoxia (8% O2 for 2 h) could induce ipsilateral cerebral HI injury in 7-day-old rat pups. To test whether the ischemia preconditioning (IP) could be established in neonatal brain in a time-dependent manner, rat pups were subjected to HI at 22 h (IP-22h group), 6 h (IP-6h group) or 2 h (IP-2h group) after reversible unilateral carotid artery ligation for 2 h. The outcome was measured by behavior assessment (P35) and pathology (P40) (percentage of brain weight reduction) and behavior (Morris water maze) on P35-P40.
Compared to the No-IP group, all the three IP groups had significantly neuroprotective effect at morphological and behavioral levels. Among the three IP groups, the IP-6h group had the worst behavioral performance and also showed more TUNEL positive cells in the cortex and hippocampus.
Twenty-four hours after HI, the reactive oxygen species (ROS) production and the expression levels of the cleavaged form of pro-apoptotic markers, such as caspase-3, caspase-8, caspase-9, poly (ADP-ribose) polymerase, and apoptosis inducing factor, were significantly lower in the IP-22h group compared to the no-IP group. In addition, the release of cytochrome c and Smac from mitochondria to cytosol after HI was also significantly reduced in the IP-22h group. In contrast, the expression of anti-apoptotic markers, such as cellular inhibitor of apoptosis-1(cIAP-1) and BCL2, but not X-linked inhibitor of apoptosis, were significantly higher in the IP-22h group. Increased expression of cIAP-1 but not activated caspase-3 was also found in the IP-22h group during preconditioning phase.
Our study suggests that but both rapid and delayed phase of IP can be established in the immature brain, suggesting the unique plasticity in the development brain. In addition, IP-mediated neuroprotective mechanisms in the immature brain involve not only the inhibition of apoptosis (caspase-dependent and caspase-independent pathways, and intrinsic and extrinsic pathways) but also increase expression of anti-apoptotic markers, such as cIAP-1. Further work will be extended to determine the role of cIAP-1 in IP and elucidate its upstream signaling during IP in the immature rat brain.

目錄

中文摘要 Ⅰ

英文摘要 Ⅲ

誌謝 Ⅴ

目錄 Ⅵ

圖表目錄 Ⅹ

第一章、緒論 1

第二章、實驗材料及實驗方法 7

第一節、實驗材料 7

1. 實驗動物 7

2. 實驗藥品與試劑 7

3. 抗體 8

4. 重要器材 9

第二節、實驗方法 10

1. 動物模式的建立 10

(1) 新生幼鼠缺血窒息腦傷模式 10

(2) 缺血前置處理模式 11

(3) 行為測試 11

(4) 形態學評估 13

2. 偵測反應性氧自由基 14

3. 西方墨點法 15

5. 免疫螢光染色 17

6. 統計分析 17

第三章、實驗結果 18

第一節、在新生鼠腦建立缺血前置處理模式 18

1. 行為測試 (Behavior test) 18

2. 形態學(Morphological)觀察 20

(1) H&E stain 20

(2) Brain weight reduction 20

(3) Nissl stain 21

(4) TUNEL stain 21

第二節、利用建立的缺血前置處理模式探討對抗缺血窒息腦傷的腦部保護的機制 22

1. 偵測反應性氧自由基(Reactive oxygen species，ROS)產量 22

2. 探討pro-apoptotic pathway 23

(1)在缺血窒息腦傷後，缺血前置處理減低新生幼鼠腦部caspase-3活化情形。 23

(2) 在缺血窒息腦傷後，缺血前置處理減少新生幼鼠腦部PARP裂解情形。 24

(3) 在缺血窒息腦傷後，缺血前置處理減少新生幼鼠腦部在extrinsic pathway中caspase-8與intrinsic pathway中caspase-9活化的情形。 24

(4) 在缺血窒息腦傷後，缺血前置處理減少cytochrome c、SMAC及AIF從mitochondria釋放到cytosol。 25

3. 探討Anti-apoptotic pathway 28

(1) 在缺血窒息腦傷後，缺血前置處理使Bcl-2在mitochondria上表現。 28

(2) 在缺血窒息腦傷後，缺血前置處理增加cIAP-1的表現，但XIAP的表現並沒有改變。 29

4. 缺血前置處理階段(preconditioning phase)機制探討 29

(1) 缺血前置處理增加cIAP-1的表現 29

(2) 缺血前置處理不會改變activated caspase-3的表現 30

5. 探討preconditioning phase可能形成腦部保護的因子 30

第四章、討論 32

第五章、參考文獻 41

第六章、圖表 47

An, H., Yu, Y., Zhang, M., Xu, H., Qi, R., Yan, X., Liu, S., Wang, W., Guo, Z., Guo, J., Qin, Z. and Cao, X. (2002) Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology, 106, 38-45.

Andreasson, K.I., Savonenko, A., Vidensky, S., Goellner, J.J., Zhang, Y., Shaffer, A., Kaufmann, W.E., Worley, P.F., Isakson, P. and Markowska, A.L. (2001) Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice. J Neurosci, 21, 8198-8209.

Benn, S.C. and Woolf, C.J. (2004) Adult neuron survival strategies--slamming on the brakes. Nat Rev Neurosci, 5, 686-700.

Blomgren, K., Zhu, C., Wang, X., Karlsson, J.O., Leverin, A.L., Bahr, B.A., Mallard, C. and Hagberg, H. (2001) Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"? J Biol Chem, 276, 10191-10198.

Boldin, M.P., Goncharov, T.M., Goltsev, Y.V. and Wallach, D. (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell, 85, 803-815.

Bolli, R. (2000) The late phase of preconditioning. Circ Res, 87, 972-983.

Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J. and Greenberg, M.E. (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96, 857-868.

Cande, C., Cohen, I., Daugas, E., Ravagnan, L., Larochette, N., Zamzami, N. and Kroemer, G. (2002) Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie, 84, 215-222.

Chan, S.L. and Yu, V.C. (2004) Proteins of the bcl-2 family in apoptosis signalling: from mechanistic insights to therapeutic opportunities. Clin Exp Pharmacol Physiol, 31, 119-128.

Chinnaiyan, A.M., O'Rourke, K., Tewari, M. and Dixit, V.M. (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 81, 505-512.

Cory, S. and Adams, J.M. (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer, 2, 647-656.

Du, C., Fang, M., Li, Y., Li, L. and Wang, X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell, 102, 33-42.

Ferriero, D.M. (2004) Neonatal brain injury. N Engl J Med, 351, 1985-1995.

Garnier, P., Ying, W. and Swanson, R.A. (2003) Ischemic preconditioning by caspase cleavage of poly(ADP-ribose) polymerase-1. J Neurosci, 23, 7967-7973.

Gewies, A. (2003) Introduction to Apoptosis. ApoReview, 1-26.

Gidday, J.M., Shah, A.R., Maceren, R.G., Wang, Q., Pelligrino, D.A., Holtzman, D.M. and Park, T.S. (1999) Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning. J Cereb Blood Flow Metab, 19, 331-340.

Ginis, I., Schweizer, U., Brenner, M., Liu, J., Azzam, N., Spatz, M. and Hallenbeck, J.M. (1999) TNF-alpha pretreatment prevents subsequent activation of cultured brain cells with TNF-alpha and hypoxia via ceramide. Am J Physiol, 276, C1171-1183.

Gong, K.Z., Zhang, Z.G., Li, A.H., Huang, Y.F., Bu, P., Dong, F. and Liu, J. (2004) ROS-mediated ERK activation in delayed protection from anoxic preconditioning in neonatal rat cardiomyocytes. Chin Med J (Engl), 117, 395-400.

Granville, D.J., Cassidy, B.A., Ruehlmann, D.O., Choy, J.C., Brenner, C., Kroemer, G., van Breemen, C., Margaron, P., Hunt, D.W. and McManus, B.M. (2001) Mitochondrial release of apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis. Am J Pathol, 159, 305-311.

Hagberg, H. (2004) Mitochondrial impairment in the developing brain after hypoxia-ischemia. J Bioenerg Biomembr, 36, 369-373.

Han, B.H., Xu, D., Choi, J., Han, Y., Xanthoudakis, S., Roy, S., Tam, J., Vaillancourt, J., Colucci, J., Siman, R., Giroux, A., Robertson, G.S., Zamboni, R., Nicholson, D.W. and Holtzman, D.M. (2002) Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem, 277, 30128-30136.

Holscher, C. (1999) Stress impairs performance in spatial water maze learning tasks. Behav Brain Res, 100, 225-235.

Hsu, H., Xiong, J. and Goeddel, D.V. (1995) The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell, 81, 495-504.

Hsu, S.Y. and Hsueh, A.J. (2000) Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm. Physiol Rev, 80, 593-614.

Hsu, Y.T., Wolter, K.G. and Youle, R.J. (1997) Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci U S A, 94, 3668-3672.

Hu, B.R., Liu, C.L., Ouyang, Y., Blomgren, K. and Siesjo, B.K. (2000) Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cereb Blood Flow Metab, 20, 1294-1300.

Hudome, S., Palmer, C., Roberts, R.L., Mauger, D., Housman, C. and Towfighi, J. (1997) The role of neutrophils in the production of hypoxic-ischemic brain injury in the neonatal rat. Pediatr Res, 41, 607-616.

Johnston, M.V., Trescher, W.H., Ishida, A. and Nakajima, W. (2001) Neurobiology of hypoxic-ischemic injury in the developing brain. Pediatr Res, 49, 735-741.

Kirino, T. (2002) Ischemic tolerance. J Cereb Blood Flow Metab, 22, 1283-1296.
Koistinaho, J., Koponen, S. and Chan, P.H. (1999) Expression of cyclooxygenase-2 mRNA after global ischemia is regulated by AMPA receptors and glucocorticoids. Stroke, 30, 1900-1905; discussion 1905-1906.

Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J. and Williams, L.T. (1997) Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science, 278, 294-298.

Liston, P., Fong, W.G. and Korneluk, R.G. (2003) The inhibitors of apoptosis: there is more to life than Bcl2. Oncogene, 22, 8568-8580.

Liu, D., Lu, C., Wan, R., Auyeung, W.W. and Mattson, M.P. (2002) Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J Cereb Blood Flow Metab, 22, 431-443.

Liu, J., Ginis, I., Spatz, M. and Hallenbeck, J.M. (2000) Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide. Am J Physiol Cell Physiol, 278, C144-153.

Merry, D.E., Veis, D.J., Hickey, W.F. and Korsmeyer, S.J. (1994) bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS. Development, 120, 301-311.

Mirjany, M., Ho, L. and Pasinetti, G.M. (2002) Role of cyclooxygenase-2 in neuronal cell cycle activity and glutamate-mediated excitotoxicity. J Pharmacol Exp Ther, 301, 494-500.

Murry, C.E., Jennings, R.B. and Reimer, K.A. (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation, 74, 1124-1136.

Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E. and Dixit, V.M. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell, 85, 817-827.

Nakai, Y., Horimoto, H., Mieno, S. and Sasaki, S. (2001) Mitochondrial ATP-sensitive potassium channel plays a dominant role in ischemic preconditioning of rabbit heart. Eur Surg Res, 33, 57-63.

Nakamura, M., Nakakimura, K., Matsumoto, M. and Sakabe, T. (2002) Rapid tolerance to focal cerebral ischemia in rats is attenuated by adenosine A1 receptor antagonist. J Cereb Blood Flow Metab, 22, 161-170.

Ohtsuki, T., Kitagawa, K., Yamagata, K., Mandai, K., Mabuchi, T., Matsushita, K., Yanagihara, T. and Matsumoto, M. (1996) Induction of cyclooxygenase-2 mRNA in gerbil hippocampal neurons after transient forebrain ischemia. Brain Res, 736, 353-356.

Otani, H. (2004) Reactive oxygen species as mediators of signal transduction in ischemic preconditioning. Antioxid Redox Signal, 6, 449-469.

Ozes, O.N., Mayo, L.D., Gustin, J.A., Pfeffer, S.R., Pfeffer, L.M. and Donner, D.B. (1999) NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature, 401, 82-85.

Rajapakse, N., Shimizu, K., Kis, B., Snipes, J., Lacza, Z. and Busija, D. (2002) Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats. Neurosci Lett, 327, 208-212.

Saito, A., Hayashi, T., Okuno, S., Ferrand-Drake, M. and Chan, P.H. (2003) Interaction between XIAP and Smac/DIABLO in the mouse brain after transient focal cerebral ischemia. J Cereb Blood Flow Metab, 23, 1010-1019.

Tanaka, H., Yokota, H., Jover, T., Cappuccio, I., Calderone, A., Simionescu, M., Bennett, M.V. and Zukin, R.S. (2004) Ischemic preconditioning: neuronal survival in the face of caspase-3 activation. J Neurosci, 24, 2750-2759.

Tomai, F., Crea, F., Chiariello, L. and Gioffre, P.A. (1999) Ischemic preconditioning in humans: models, mediators, and clinical relevance. Circulation, 100, 559-563.

Vanden Hoek, T.L., Becker, L.B., Shao, Z., Li, C. and Schumacker, P.T. (1998) Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem, 273, 18092-18098.

Vannucci, R.C. (1990) Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage. Pediatr Res, 27, 317-326.

Vannucci, R.C., Connor, J.R., Mauger, D.T., Palmer, C., Smith, M.B., Towfighi, J. and Vannucci, S.J. (1999) Rat model of perinatal hypoxic-ischemic brain damage. J Neurosci Res, 55, 158-163.

Vekrellis, K., McCarthy, M.J., Watson, A., Whitfield, J., Rubin, L.L. and Ham, J. (1997) Bax promotes neuronal cell death and is downregulated during the development of the nervous system. Development, 124, 1239-1249.

Vincenz, C. and Dixit, V.M. (1997) Fas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J Biol Chem, 272, 6578-6583.

Xuan, Y.T., Tang, X.L., Banerjee, S., Takano, H., Li, R.C., Han, H., Qiu, Y., Li, J.J. and Bolli, R. (1999) Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res, 84, 1095-1109.

Yue, X., Mehmet, H., Penrice, J., Cooper, C., Cady, E., Wyatt, J.S., Reynolds, E.O., Edwards, A.D. and Squier, M.V. (1997) Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia-ischaemia. Neuropathol Appl Neurobiol, 23, 16-25.

Zhu, C., Wang, X., Hagberg, H. and Blomgren, K. (2000) Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia. J Neurochem, 75, 819-829.