臺灣博碩士論文加值系統

背景：對於創傷性腦損傷(TBI)患者的神經系統後遺症的治療處理及復健一直是很困難而緩慢的, 並且也是醫療團隊的一大挑戰。儘管現在科技進步,我們仍無法擁有令人滿意的治療結果. 許多在基礎實驗模型中證明有療效的藥物常無法在臨床上有效治療TBI患者，許多的評論對於動物模型中顯示有療效的藥物能否在臨床上應用開始持疑。在這方面, 對於能更細部規劃和準確控制的TBI動物模型,合併多領域及多重機轉研究的方法學, 並為以後的療效評估建立明確的處理標準, 成為前臨床研究中基礎研究的重要課題之一, 頭部外傷後主要的病理機轉包括自由基釋放造成氧化壓力，興奮性毒素的作用及發炎反應等，這些機制交互作用會對神經細胞造成後續的二度傷害。而若想要達到神經保護功效，可使用多重機制阻斷的合併治療方式。本研究選用了黃芩素(Wogonin), 探討其於實驗性腦創傷神經保護的功效及保護機制。Wogonin是萃取自中草藥黃芩的主要類黃酮素，在實驗中顯示具有抗氧化及抗發炎雙重機制，我們將研究Wogonin對TBI小鼠的影響, 包含抗發炎分子的量測，功能和組織學, 腦水腫的影響，以及TLR4/ NF-kappa B相關的信號傳導路徑的探討。
方法/結果：將接受腦皮質撞擊傷(CCI)的小鼠於10分鐘後進行功能及組織學，血腦屏障（BBB）的通透性和腦水含量進行測量，評估。 TLR4/ NF-kappa B相關的炎症分子也一併量測。Wogonin治療組顯著提高功能恢復和減少挫傷體積。Wogonin也顯著減少神經元死亡，血腦屏障通透性和腦水腫。這些變化包含顯著減少的白血球細胞浸潤，小膠質細胞活化，TLR4的表現，NF-kappa B結合活性，基質金屬蛋白酶9的活性和表達的炎症介質，包括白細胞介素1，白細胞介素-6，巨噬細胞炎性蛋白-2，和COX-2。
討論：我們的研究結果顯示，在TBI小鼠傷後使用Wogonin治療能改善長期功能和組織學的結果，降低腦水腫，減弱了TLR4/ NF-kappa B -介導的炎症反應。漢黃芩素的神經保護作用可能與TLR4/ NF-kappa B信號傳導路徑的調節有關。另一方面，我們展示了這一個基於腦損傷皮質撞擊損傷模型（CCI）的療效評估和機轉探索系統.。我們使用了三個不同方向的評估測試，包括：（1）功能評測（2）解剖學和組織學評估（3）生物分子量測, 並整合至我們的平台, 並將此CCI損傷模型以綜合性，多機制的流程，進行了wogonin的實驗, 以說明此平台的可行性和實用性。進一步的, 我們希望以CCI腦損傷模式來擴大我們的研究, 例如不同藥物及不同機轉的神經保護作用，甚至神經再生領域,例如細胞療法等; 另外也希望整合內文中提到的新技術和新設備，因為具有即時採集，非破性量測或最少的樣品要求，在活細胞或組織的操作和節省時間，低耗材的實際需求等特性, 可望能在未來神經科學的研究中提供新的進展及應用。

Background: Treating and managing neurological sequelae of traumatic brain injury (TBI) patients are difficult and keep challenging medical stuff despite of the advancement of technology. Many drugs that proved efficacy in experimental models failed to show the benefits in the treatment of TBI population, so as many reviews did not support that those efficacy treatment in animal models effected. There is strong need of well-designed and controlled animal models with an integrative, multi-mechanism approach for drug discovery and study in order to establish definitive treatment standards for this patient population;
For that a proper understanding of animal trials requires that diverse aspects and mechanisms of the injury be taken into account, and it is well studied that TBI initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment, this study was undertaken to investigate the effects of wogonin, a flavonoid with potent anti-inflammatory properties, on functional and histological outcomes, brain edema, and toll-like receptor 4 (TLR4)- and nuclear factor kappa B (NF-kappa B)-related signaling pathways in mice following TBI.
Methodology/Principal Findings: Mice subjected to controlled cortical impact injury were injected with wogonin 20, 40, or 50 mg‧kg-1) or vehicle 10 min after injury. Behavioral studies, histology analysis, and measurement of blood-brain barrier (BBB) permeability and brain water content were carried out to assess the effects of wogonin. Levels of TLR4/NF-kappa B-related inflammatory mediators were also examined. Treatment with 40 mg‧kg-1 wogonin significantly improved functional recovery and reduced contusion volumes up to post-injury day 28. Wogonin also significantly reduced neuronal death, BBB permeability, and brain edema beginning at day 1. These changes were associated with a marked reduction in leukocyte infiltration, microglial activation, TLR4 expression, NF-kappa B binding activity, matrix metalloproteinase-9 activity, and expression of inflammatory mediators, including interleukin-1beta, interleukin-6, macrophage inflammatory protein-2, and cyclooxygenase-2.
Conclusions/Significance: Our results show that post-injury wogonin treatment improved long-term functional and histological outcomes, reduced brain edema, and attenuated the TLR4/NF-kappa B-mediated inflammatory response in mouse TBI. The neuroprotective effects of wogonin may be related to modulation of the TLR4/NF-kappa B signaling pathway. On the other hand, we have set up an efficacy evaluation system and mechanism exploration system of therapeutic agents for TBI by experimental animal brain injury model- controlled cortical impaction (CCI) injury model. Three major groups of evaluation tests including: (1) functional evaluation (2) anatomical and histological evaluation (3) Biomolecular evaluation are introduced and integrated into our platform. And we also have demonstrated the feasibility and practicability of our CCI injury model with an integrative, multi-mechanism approach through the experiment of wogonin. Prospectively, with this practable CCI model we are capable to expand our research to different fields of neuroprotection, or neuroregeneration like cell therapies; also the integration of the new technology and equipment mentioned in contents, which are well characterized with real-time acquisition, non-sample destruction or minimal sample requirement, operation in living cell or tissue; and the practical benefits of time-saving, less consumables requirement, would make great progression of research in neuroscience .

CATALOG
口試委員會審定書 I
ACKNOWLEDGEMENTS III
中文摘要 V
ABSTRACT VII
FIGURES XVII
TABLES XIX

CHAPTER 1 INTRODUCTION 1
1.1 Traumatic Brain Injury (TBI) 1
1.2 Injury Pathophysiology 3
1.2.1 Review of Primary Injury in Cellular Level 5
1.2.2 Review of Secondary Injury in Cellular Level 6
1.3 Pharmacotherapy in preventing secondary injury 8
1.4 Mechanism exploration 10
1.5 Introduction of Wogonin 12

CHAPTER 2 REVIEW OF ANIMAL MODELS 17
2.1 Summary of in vivo Experimental Models 19
2.2 Weight-Drop Injury (WDI) Model 21
2.3 Fluid Percussion Model 24
2.4 Impact Acceleration Model 26
2.5 Controlled Cortical Impact (CCI) Model 27
2.5.1 History and Basic Introduction 27
2.5.2 Physiological Exploration in CCI Model 31
2.5.3 Hardware Setup 32
2.6 Summary of TBI animal models 35

CHAPTER 3 MATERIALS AND METHODS 37
3.1 Surgical Procedures 37
3.2 Experimental Protocol and Dose Selection 39
3.2.1 Neurological Functional Evaluation 40
3.2.2 Evaluation of Metabolic Characteristics 42
3.2.3 Tissue Processing and Histology 43
3.2.4 Contusion Volume and hemispheric Enlargement Analysis 43
3.2.5 Evaluation of Blood-Brain Barrier Permeability 44
3.2.6 Brain Water Content 45
3.2.7 FJB Histochemistry 45
3.2.8 TUNEL Staining 46
3.2.9 Immunohistochemistry 46
3.2.10 Quantification of FJB, TUNEL, MPO, and Iba-1 Staining 48
3.2.11 Western Blot Analysis 49
3.2.12 Enzyme-Linked Immunosorbent Assay 50
3.2.13 Gelatin Zymography 51
3.2.14 Real-time Quantitative RT-PCR 51
3.2.15 Electrophoretic Mobility Shift Assay 52
3.2.16 Statistical Analyses 54

CHAPER 4 RESULTS 55
4.1 Metabolic Characteristics 55
4.2 Post-Injury Wogonin Administration Improves Neurobehavioral Recovery 55
4.2.1 Rotarod Test 55
4.2.1 Modified Neurological Severity Score 56
4.2.3 Beam Walk Test 57
4.3 Post-Injury Wogonin Administration Reduces Cortical Contusion Volume, Blood-Brain Barrier Permeability, and Brain Edema 60
4.4 Post-Injury Wogonin Administration Reduces Neuronal and Apoptotic Cell Death 64
4.5 Post-Injury Wogonin Administration Reduces Neutrophil Infiltration and Microglial Activation 67
4.6 Post-injury Wogonin Administration Reduces IL-1β, IL-6, MIP-2, and COX-2 Expression, but has no Effect on MCP-1 Expression 70
4.7 Post-Injury Wogonin Administration Reduces MMP-9 Enzymatic Activity 73
4.8 Post-Injury Wogonin Administration Reduces NF- kappa B Binding Activity 73
4.9 Post-Injury Wogonin Administration Reduces TLR-4 Protein Expression 73

CHAPTER 5 DISCUSSION AND CONCLUSION 79

CHAPTER 6 PROSPECTIVE IN METHODOLOGY 87
6.1 Animal Models in Preclinical Evaluation of Neuroprotective Drugs 87
6.2 Evaluation Methods in Preclinical Evaluation of Neuroprotective Drugs 90
6.2.1 Morphology 92
6.2.2 Histology 95
6.2.3 Extractant Molecular Analysis 96
6.3 Prospective of Technic and Equipment in Wogonin Study 100
6.3.1 Introduction of MRI / DCE-MRI 100
6.3.2 Two photon Laser Excitation Microscopy (TPEM) 102
6.3.3 Surface Plasma Resonance (SPR) biosensor 105
6.4 Conclusion 110

Appendix I
FDA Protocol for Drug Development 129

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