臺灣博碩士論文加值系統

脊髓損傷是導致肢體癱瘓及器官功能缺損的重大傷害。外力直接撞擊脊髓造成出血以及 組織破壞等稱之為初級傷害。接續的發炎反應、氧化性壓力以及細胞凋亡持續地破壞脊 髓,這個現象稱為次級傷害。近年來,脊髓損傷的治療大多著重在降低次級傷害的發生, 期望可以增進肢體運動功能的回復。過去的研究發現,腺苷接收器在脊髓損傷所誘發的 發炎反應中,扮演著重要的調控角色。咖啡因不只是一種被廣為使用的精神刺激物,同 時也是一種非選擇性的腺苷接收器抑制劑。在腦部損傷的動物實驗中發現,咖啡因可透 過抗氧化等作用保護神經,但是咖啡因對於脊髓損傷後自發性肢體運動功能回復的影響, 則尚未清楚。本研究採用脊髓損傷撞擊器(NYU MASCIC impactor)撞擊大鼠脊髓第九胸 椎處來建立損傷之動物模式。手術後連續七天在腹腔注射咖啡因(30 mg/Kg)。實驗對 照組則注射磷酸緩衝生理食鹽水。本實驗以肢體功能計量表(BBB rating scale)每週一次 連續四週評估後肢運動功能。結果發現,此一劑量咖啡因不會影響生理參數,但卻抑制 自發性的肢體運動功能回復。在最後一次功能評估後,立刻取出脊髓進行組織型態分析。 結果發現,咖啡因(1)降低脊髓組織剩餘量,(2)增加去髓鞘化面積,(3)促進星狀細胞及 微膠細胞的活化,(4)增加脊髓內神經細胞的死亡,以及(5)減少神經纖維的含量。在另 一組實驗中,我們在大鼠脊髓損傷後,連續腹腔給予咖啡因(30 mg/Kg)七天,接著將 其脊髓取出運用西方墨漬法分析數種蛋白質的表現量。結果顯示,咖啡因(1)抑制腺苷接 收器A2a以及A3的表現、(2)抑制細胞生長存活的訊息傳導因子(P-Akt、p-ST A T3及p-Erk) 的活化,(3)增加氧化壓力,以及(4)提升自噬現象及細胞凋亡的產生。總結本研究,脊 髓損傷後咖啡因會抑制自發性肢體功能的回復;此一現象可能是透過腺苷接受器抑制細 胞生長存活因子的表現,增加細胞氧化壓力,自噬現象,以及細胞凋亡等現象,進而導 致神經性發炎與脊髓的去髓鞘化,最終使得次級傷害更加嚴重。有鑒於此劑量咖啡因在 脊髓損傷所帶來的負面影響,此類患者應避免投予含有咖啡因的藥物以及咖啡因的過量攝取。

Spinal cord injury (SCI) results in paralysis and organ impairment. The pathology of SCI includes primary and secondary injury that is involved in neurological dysfunction. Primary injury of SCI refers to an injury that is directly caused by a physical or chemical insults; whereas, secondary injury refers to injuries that are caused by primary injury-induced subsequent events, such as inflammation, oxidative stress and apoptosis. SCI therapy has been focused on preventing secondary injury to reduce lesion area so that functional outcome after a SCI may be improved. Adenosine receptors (ARs) are a major regulator of inflammation after SCI. Caffeine, one of the most widely used psychoactive substances, acts as a non-selective AR blocker and has neuroprotective effects in brain injury. Nevertheless, the effect of caffeine on spontaneous motor function recovery after a SCI remains unknown. The contusion SCI animal model was generated by hitting the thoracic 9th spinal cord level using an impactor (NYU MASCIC impactor). Caffeine (30 mg/Kg) was injected intraperitoneally (i.p.) for 7 days after SCI immediately, while the control group was injected with PBS. Hindlimb motor function was evaluated by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale weekly after SCI for 4 weeks. Initial test showed that 30 mg/Kg of caffeine treatment did not affect body weight or hindlimb motor function in normal rats. However, caffeine (30 mg/Kg) treatment reduced the BBB scores compared to the PBS treatment group after SCI. Spinal cord was collected after the final behavior test for morphological analyze. The results showed that caffeine (1) decreased spinal cord tissue sparing area, (2) decreased the myelinated area, (3) promoted astrocyte and microglia activation, (4) triggered neuron necrosis, and (5) decreased the quantity of neurofilaments. In another experiment, rats were daily injected with caffeine 30mg/Kg, i.p. for 7 days, and the spinal cords were collected to analyze protein expression level by Western blot analysis. The results showed that caffeine decreased levels of adenosine receptor, A2a and A3, and survival signaling molecules including p-Akt, p-Erk and p-STAT3. Meanwhile, caffeine increased levels of Nrf2, LC3II and the cleavage form of caspase-3. These results suggested that caffeine treatment inhibits spontaneous motor function recovery through ARs by increasing oxidative stress, decreasing survival signal pathways, and triggering autophagic related cell death. The caffeine-induced inhibition of neural repair and promotion of neuroinflammation may aggravate the secondary injury. Accordingly, these findings suggest that the SCI patients should avoid high dose of caffeine ingestion.

CONTENTS
FIGURE LIST
中文摘要 1
ABSTRACT 2
ABBREVIATIONS 3

1 INTRODUCTION 4
1.1 SPINAL CORD INJURY 5
1.2 OXIDATIVE STRESS 5
1.3 AUTOPHAGY 6
1.4 ADENOSINE RECEPTOR 7
1.5 CAFFEINE 9
1.6 THE AIM OF THIS STUDY 10

2 EXPERIMENTAL DESIGN 11
2.1 THE EFFECT OF CAFFEINE ON MOTOR FUNCTION RECOVERY AFTER SCI 12
2.2 THE EFFECT OF CAFFEINE ON MORPHOLOGICAL CHANGES AFTER SCI 13
2.3 THE EFFECT OF CAFFEINE ON PROTEIN EXPRESSION 14

3 MATERIALS AND METHODS 15
3.1 ANIMALS 16
3.2 CONTUSION SPINAL CORD INJURY (SCI) MODEL 16
3.3 DRUG ADMINISTRATION 16
3.4 BASSO, BEATTIE, BRESNAHAN LOCOMOTOR RATING SCALE 17
3.5 PERFUSION, FIXACTION AND SECTION 17
3.6 MEASUREMENT OF SPARED MYELIN IN WHITE MATTER AFTER SCI 18
3.7 MEASUREMENT OF CELL NECROSIS IN SPINAL CORD AFTER SCI 18
3.8 MEASUREMENT OF TISSUE SPARING AFTER SCI 19
3.9 MEASUREMENT OF THE MICROGLIA NUMBER AFTER SCI 19
3.10 MEASUREMENT OF GLIOSIS AFTER SCI 20
3.11 MEASUREMENT OF NEUROFILAMENT AFTER SCI 20
3.12 WESTERN BLOT 21
3.13 STATISTICS 22

4 RESULTS 23
5 DISCUSSION 29
6 CONCLUSION 36
7 REFERENCES 38
8 FIGURES 53
9 ACKNOWLEDGEMENT 74
10 PUBLICATION LIST 76
11 PUBLICATIONS 79

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