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研究生:謝育鳴
研究生(外文):Yu-Ming Hsieh
論文名稱:高壓氧對於中大腦動脈阻塞大鼠神經保護機制之探討
論文名稱(外文):Neuroprotection of hyperbaric oxygen in the transient focal middle cerebral artery occlusion rats
指導教授:楊雅如楊雅如引用關係
指導教授(外文):Yea-Ru Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:物理治療暨輔助科技學系
學門:醫藥衛生學門
學類:復健醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:53
中文關鍵詞:高壓氧腦源性神經營養因子硫氧化還原蛋白缺血性腦損傷
外文關鍵詞:Hyperbaric oxygenBrain-derived neurotrophic factorThioredoxinCerebral ischemia
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研究背景與目的:缺血性腦中風的發生會導致腦部氧氣濃度劇烈減少,進而使受影響區域的腦細胞死亡,其中包含細胞壞死(necrosis)及細胞凋亡(apoptosis)。因此,在腦部發生缺血後的治療,立即提供充足氧氣給予受影響的腦部組織,遂成為拯救凋亡細胞的重要治療策略之一。先前已有研究顯示,利用高壓氧預處理(HBO preconditioning)於全腦缺血動物模型(global cerebral ischemia)後,能夠有效抑制腦部細胞凋亡、提升腦源性神經營養因子(BDNF)、及減低p38 MAPK活性。此外,另有研究發現,於中大腦動脈阻斷(MCAO)後,屬於抗氧化因子之一的硫氧化還原蛋白(Trx)在缺血區域周圍的濃度有顯著提升,且對於降低大腦缺血後之梗塞體積有所影響,因此,硫氧化還原蛋白被認為具有減少大腦缺血性傷害的保護效應。本研究之主要目的為探討暫時性中大腦動脈阻塞後,腦源性神經營養因子及硫氧化還原蛋白於高壓氧介入抑制細胞凋亡效應之功能。研究方法:50隻Sprague-Dawley雄性大鼠隨機分配至模擬手術組(sham group,n = 14)、對照組(MCAO group,n = 18)及高壓氧介入組(MCAO + HBO group,n = 18)。分配至對照組及高壓氧介入組的大鼠將經歷暫時性中大腦動脈阻斷一小時,隨後鬆開阻斷處,使血管血液能重新灌流。待血液灌流後三小時,高壓氧介入組之大鼠將被放入高壓氧艙槽,接受3個絕對大氣壓(ATA)及100%氧氣濃度之治療一小時。為探討高壓氧之效應,所有大鼠於血液灌流後24小時被犧牲,並且利用TUNEL、ELISA與western blotting分別量測凋亡細胞比例、腦源性神經營養因子濃度與硫氧化還原蛋白表現量。利用單因子變異數分析(One-way ANOVA)比較三項結果於各組間的差異,並以Bonferronitest進行事後檢定。顯著差異定為P<0.05。研究結果:大腦缺血後,受損側皮質的細胞凋亡比例大幅增加約60%(P<0.01),但接受高壓氧介入後,細胞凋亡比例則明顯減少約40%(P<0.01)。高壓氧介入組腦皮質中的腦源性神經營養因子濃度相較對照組亦有顯著增加(P<0.05)。此外,大腦缺血後,藉由高壓氧介入能夠增加約55%硫氧化還原蛋白的表現(P<0.01)。結論:高壓氧介入能夠促使腦源性神經營養因子濃度和硫氧化還原蛋白表現量增加,此二者可能調控下游的訊息傳遞路徑以減少因缺血引起的腦部細胞凋亡。臨床意義:本研究成果對於高壓氧在缺血性中風病人之臨床應用將有所助益。
Background and Purpose: Ischemic stroke resulted in dramatic reduction of oxygen level in the brain and subsequent cell death, including necrosis and apoptosis. The acquisition of abundant oxygen supplied to affected brain tissue was one of the most important treatment strategies for rescuing apoptosis in the ischemic brain. A previous study showed that hyperbaric oxygen (HBO) preconditioning could inhibit apoptosis, promote brain-derived neurotrophic factor (BDNF), and inactivate p38 MAPK in the brain after global cerebral ischemia. Additionally, other studies revealed an antioxidant factor, thioredoxin (Trx), increased in the ischemic penumbra and could decrease the infarct volume following cerebral ischemia. The purpose of present study was to clarify the role of BDNF and Trx on post-ischemic HBO intervention induced neuroprotection in transient MCAO rats.
Methods: Fifty male Sprague-Dawley (SD) rats were randomly allocated to the sham group (n = 14), MCAO group (n = 18), and MCAO + HBO group (n = 18). The rats in the latter two groups underwent the right middle cerebral arteries were blocked for 1 hour and then reperfused. Until 3 hours following the beginning of reperfusion, the rats in MCAO + HBO group were placed into a HBO chamber for 1 hour and receive 100% oxygen at 3 ATA. All rats were sacrificed at 24 hours after the onset of reperfusion for investigating the effects of HBO intervention. The apoptosis in the ischemic area was evaluated with terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL). The BDNF level was evaluated with Enzyme-Linked ImmunoSorbent Assay (ELISA) and the Trx level was measured by western blotting. One-way ANOVA was used to compare the difference of apoptosis, BDNF level, and the expression of Trx among three groups. The Bonferroni test was used as the post hoc test. P value < 0.05 was regarded as statistically significant.
Results: The results demonstrated that after cerebral ischemia, the apoptosis in the ipsilesional cortex was increased (TUNEL-positive cells/Hoechst: Sham 6.59±0.32% versus MCAO 71.67±1.43%, p<0.01). However, following the treatment with HBO, the apoptosis was significantly inhibited (MCAO 71.67±1.43% versus MCAO+HBO 32.42±0.68%, p<0.01). The BDNF level was significantly promoted in the ipsilesional cortex of MCAO rats after receiving HBO (MCAO+HBO 1.61±0.11pg/mg versus MCAO 0.75±0.05pg/mg, p<0.05). Furthermore, the HBO intervention induced the expression of Trx after cerebral ischemia (Trx/β-actin: MCAO+HBO 125.44±6.11% versus MCAO 70.14±7.12%, p<0.01).
Conclusion: HBO intervention enhanced the BDNF level and the expression of Trx that may subsequently regulate the downstream signaling pathways to decrease apoptosis in the brain of rats with MCAO.
Clinical Relevance: Our study results would be beneficial to the clinical HBO application on ischemic stroke patients.
Thesis Approval Form i
Chinese AbstracT ii
English Abstract iv
Table of Contents vi
List of Figures ix
List of abbreviations x
Chapter 1 Introduction 1
1.1 Background and Motivation 1
1.2 Purposes 2
1.3 Research Hypothesis 3
1.4 Significance of Study 3
Chapter 2 Literature Review 4
2.1 Pathophysiology of cerebral ischemia 4
2.2 The events of apoptosis in cerebral ischemia 5
2.3 Hyperbaric oxygen 6
2.3.1 Introduction of hyperbaric oxygen 6
2.3.2 Effects of hyperbaric oxygen on cerebral ischemia 7
2.3.3 Potentially harmful effects of hyperbaric oxygen 8
2.4 Brain-derived neurotrophic factor and cerebral ischemia 10
2.5 Oxidative stress after cerebral ischemia 11
2.5.1 Reactive oxygen species 11
2.5.2 Redox systems 12
2.5.3 Thioredoxin as a regulator of apoptosis 13
2.6 Summary 14
Chapter 3 Materials and Methods 16
3.1 Animals 16
3.2 Animal groups and experimental protocol (Figure 1) 16
3.3 Induction of transient focal middle cerebral artery occlusion 17
3.4 Hyperbaric oxygen intervention 17
3.5 TUNEL staining for measuring apoptosis 17
3.6 ELISA method for detection of brain-derived neurotrophic factor 18
3.7 Western blotting for detection of Thioredoxin 19
3.8 Statistical analysis 20
Chapter 4 Results 21
4.1 Hyperbaric oxygen intervention decreased apoptosis after cerebral ischemia 21
4.2 Hyperbaric oxygen intervention increased brain-derived neurotrophic factor level after cerebral ischemia 21
4.3 The expression of thioredoxin was promoted by hyperbaric oxygen intervention following cerebral ischemia 22
Chapter 5 Discussion 23
5.1 Effects of hyperbaric oxygen on apoptosis after cerebral ischemia 23
5.2 The role of brain-derived neurotrophic factor 25
5.2.1 Brain-derived neurotrophic factor was reduced following cerebral ischemia 25
5.2.2 Brain-derived neurotrophic factor was induced by hyperbaric oxygen intervention after cerebral ischemia 25
5.2.3 Increased brain-derived neurotrophic factor might trigger the downstream survival pathways 26
5.3 The expression of thioredoxin was elicited by hyperbaric oxygen intervention after cerebral ischemia 27
5.4 Possible interaction between survival pathway and apoptotic pathway 28
5.5 Parameters of hyperbaric oxygen intervention and others 29
5.6 Limitation of present study 30
5.7 Relevant research in the future 31
Chapter 6 Conclusions 32
References 33
Appendix 53

LIST OF FIGURES

Figure 1. Experimental procedure 45
Figure 2. HBO chamber (UO 300AR, United Oxygen Biotech, inc., Taiwan) 46
Figure 3. Cellular apoptosis in the cortex after cerebral ischemia 47
Figure 4. The differences of cellular apoptosis among sham, MCAO, and MCAO+HBO groups 48
Figure 5. Merge the results of TUNEL-positive cells and Hoechst staining 49
Figure 6. Quantification of apoptotic cells in the ischemic region 50
Figure 7. The comparison of BDNF level in ipsilesional frontal and parietal cortex among groups 51
Figure 8 (a, b). The comparison of the expression of Trx in ipsilesional frontal and parietal cortex 52
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