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研究生:陳敞牧
研究生(外文):Chang-Mu Chen
論文名稱:研究美滿庭、茶多酚及厚朴酚對各式鼷鼠興奮性神經毒性的保護機制
論文名稱(外文):Studies on the neuroprotective effects of memantine, tea polyphenol, and honokiol in various mouse excitotoxic models
指導教授:蕭水銀蕭水銀引用關係
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
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:125
中文關鍵詞:厚朴酚鉀腺嘌呤核甘三磷酸水解酶突觸體反應性氧化物粒線體膜電位粒線體還原酶中大腦動脈阻塞NMDA受體美滿庭茶多酚移動行為測驗鎂腺嘌呤核甘三磷酸水解酶鈣離子興奮性神經毒性腦室癲癇攀爬測驗滾筒式跑步機平衡測驗磷脂酶A2β-雨傘節毒素水迷宮十字迷宮
外文關鍵詞:honokiolNa+K+-ATPasesynaptosomesreactive oxygen speciesmitochondrial membrane potential(△Ψm)mitochondrial methylthiazoletetrazolium (MTT) reductasemiddle cerebral artery occlusionNMDA receptormemantinetea polyphenollocomotor activityMg2+-ATPaseintrasynaptosomal Ca2+ concentrationexcitotoxicitycerebral ventricleseizureclimbing testrotarodPLA2β-bungarotoxinwater maxeplus maze
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在工業化國家中,腦中風已經名列十大死因的第三位。其發生率為每十萬人中,約有250-400個病例,而死亡率則高達三分之一。由於腦組織會大量地消耗氧氣及葡萄糖,而且其能量的產生幾乎全靠氧化磷酸化反應(oxidative phosphorylation),因此,如果腦組織的局部血流受阻而導致氧氣及葡萄糖的運送缺乏,便會使能量製造不足而無法維持正常的離子濃度差度(ion gradients)。所以,隨著能量耗盡,膜電位差變小,造成神經元以及膠原細胞膜去極化(membrane depolarization),引發鈣離子通道(calcium channels)被活化,另外,興奮性胺基酸(excitatory amino acids)的釋出,促使NMDA受體以及其它穀氨酸(glutamate)受體的活化,而使神經細胞內鈣離子濃度過高,進而活化磷脂酶A2(phospholipase A2)及環氧酶(cyclooxygenase),產生超過體內代謝能力的自由基造成脂肪過氧化(lipid peroxidation)以及細胞膜的損害。這種所謂腦缺血再灌流所產生的神經傷害的作用機制,在其他腦傷(brain trauma)、甚至於各種不同的神經退化症,也都認為具有此種共同的神經傷害機制。於是針對此作用機制,許多的研究指向NMDA受體拮抗劑或抗氧化劑之研究,似乎具有預防或治療這些腦部疾病的潛力,本論文的目的在於建立各種鼷鼠興奮性神經毒性模式,以便於研究開發各種可能具有保護神經免於傷害的藥物(厚朴酚、茶多酚及美滿庭),有關各種實驗模式及藥物作用機轉分述如下:
壹、厚朴酚(honokiol)可緩解缺血小鼠腦部下降的鈉、鉀ATPase(Na+,K+-ATPase)活性及粒線體膜電位(mitochondrial membrane potential),並減少反應性氧化物(reactive oxygen species)的產生
厚朴酚為中藥厚朴(Magnolia officinalis)的萃取物,在過去的文獻已被證實具有抗氧化、抗發炎、以及抗焦慮等藥理作用,此外,它還具有增加癲癇發作的閥值 (seizure threshold)、以及促進神經生長等作用。本實驗證實厚朴酚對腦部缺血具有神經保護作用。在實驗中,我們將厚朴酚經腹腔注入鼷鼠(ICR mouse)體內,時間點分別是在鼷鼠中大腦缺血之前十五分鐘以及之後六十分鐘給藥。結果發現:腦部缺血會造成突觸體(synaptosome)的反應性氧化物生成增加、粒線體膜電位(△Ψm)下降、以及MTT還原反應下降,同時,也會造成鈉、鉀腺嘌呤核甘三磷酸水解酶(Na+, K+-ATPase)活性下降。使用厚朴酚則不但可以顯著減少腦梗塞的體積及突觸體的反應性氧化物增加,而且也顯著減少粒線體膜電位下降、MTT還原反應下降、以及鈉、鉀腺嘌呤核甘三磷酸水解酶活性下降的程度。因此,我們發現厚朴酚具有保護腦缺血再灌流(ischemia-reperfusion)傷害的能力。由於腦部缺血是工業化國家的主要死因之一,厚朴酚作為腦部缺血的神經保護藥物具有臨床應用的潛力,得作進一步的研究。
貳、探討合併使用美滿庭(memantine)以及茶多酚(tea polyphenol)對鼷鼠興奮性神經毒性模式(excitotoxicity)的保護機轉
NMDA受體相當廣泛地分佈在腦部,它的作用是傳導腦部的興奮性訊息,因此對於腦部功能的正常運作十分重要。然而,過多的穀氨酸鹽會造成NMDA受體的過分刺激,結果導致細胞內鈣離子的濃度上升以及興奮性毒性。長久以來,由於粒腺體功能喪失所伴隨的鈣離子濃度平衡失調,以及細胞內氧化壓力(oxidative stress)的上升,都被認為是興奮性神經毒性造成細胞傷害的主要原因。我們在本實驗中,探求在鼷鼠的興奮性神經毒性模式中,單獨使用或是併用美滿庭以及茶多酚的神經保護效果。我們將美滿庭(10 mg/kg/day)以及茶多酚(60 mg/kg/day)(單獨使用或是併用),在施行鼷鼠的興奮性神經毒性手術的前兩天,連續經口灌食。手術當天,我們將0.3微升(μl)的NMDA(335mM, pH7.2)注射到鼷鼠左側的紋狀體。至於鼷鼠的移動行為測驗(locomotor activities),則在手術前、以及手術24小時後進行。作完移動行為測驗之後,我們將鼷鼠犧牲,取得突觸體(synaptosome)以便做後續的神經生化實驗。結果顯示,單用美滿庭可以在鼷鼠興奮性神經毒性模式中,藉由減少粒線體膜電位(△Ψm)以及還原酶(reductase)活性的下降來保留粒線體的功能,此外,也可以減少鈣離子濃度的上升。而單用茶多酚則可藉由減少突觸體(synaptosome)反應性氧化物(reactive oxygen species)的產生,來減少鈉、鉀腺嘌呤核甘三磷酸水解酶以及鎂腺嘌呤核甘三磷酸水解酶(Mg2+-ATPase)的功能下降。然而,單獨使用美滿庭或是茶多酚都無法促使因興奮性神經毒性所造成的運動功能(locomotor activity)受損有所改善。如果將美滿庭以及茶多酚合併使用,則不但可以減少粒線體膜電位(△Ψm)以及還原酶(reductase)活性的下降來保留粒線體的功能以及減少鈣離子濃度的上升,也能減少突觸體反應性氧化物的產生,來減少鈉、鉀腺嘌呤核甘三磷酸水解酶以及鎂腺嘌呤核甘三磷酸水解酶的功能下降。更重要的是,合併使用美滿庭以及茶多酚可以改善因興奮性神經毒性造成運動功能的受損。因此,合併使用美滿庭及茶多酚比單用美滿庭或茶多酚在鼷鼠的興奮性神經毒性模式下,更具有神經保護的能力。這樣的研究結果使得使用美滿庭及茶多酚在一些臨床上與興奮性神經毒性有關的疾病上,如腦部創傷、腦部缺血、癲癇、阿茲海默症(Alzheimer’s disease)等,有可以應用的潛力存在。
参、建立一種新的鼷鼠興奮性神經毒性模式並探討植物性多酚(phytophenols)及美滿庭之神經保護作用
興奮性神經毒性一向被認為與多種神經疾病有關,在本實驗中,我們試著建立一種新的興奮性神經毒性動物模式,以便利用這種動物模式來研究開發對興奮性神經毒性的有效治療藥物。我們利用立體定位儀,將3微升(μl)NMDA(7mM, pH7.2)注入鼷鼠(ICR)的腦室裡。我們將美滿庭(10 mg/kg/day)、茶多酚(60 mg/kg/day)以及厚朴酚(1 mg/kg/day)(單獨使用或是併用),在施行鼷鼠的興奮性神經毒性手術之前,連續經口灌食兩天。鼷鼠在接受NMDA注射後,很快就會有癲癇發作,接著在24小時後,我們進行移動行為測驗、攀爬測驗(climbing test)以及滾筒式跑步機平衡測驗(motor equilibrium performance on rotarod)等神經行為測驗,發現都有變差的情形。隨後我們將鼷鼠犧牲,取出腦組織進行神經生化實驗,發現NMDA不但會造成突觸體反應性氧化物的產生以及鈣離子的濃度增加,也會使得粒線體膜電位、還原酶、鈉、鉀腺嘌呤核甘三磷酸水解酶以及鎂腺嘌呤核甘三磷酸水解酶的活性下降。我們藉由這些因興奮性神經毒性造成的傷害嚴重程度,可用來評估上述藥物的神經保護效果。結果發現,單獨使用厚朴酚、合併使用茶多酚和美滿庭以及合併使用厚朴酚和美滿庭都有顯著的神經保護效果,包括減少大發作(generalized seizure)的發生、減少癲癇分數(seizure score)、延長打藥到癲癇發作的時間(time latency)、減少移動行為測驗、攀爬測驗以及滾筒式跑步機平衡測驗等神經行為測驗上的異常表現等。另外,使用上述藥物也可減少突觸體反應性氧化物的產生、減少鈣離子濃度的上升、減少粒線體膜電位(△Ψm)以及還原酶(reductase)活性的下降以及減少鈉、鉀腺嘌呤核甘三磷酸水解酶以及鎂腺嘌呤核甘三磷酸水解酶的功能下降。因此,我們利用NMDA注入鼷鼠腦室而建立起一個新的興奮性神經毒性動物模式,並藉以測試出單獨使用厚朴酚、合併使用茶多酚和美滿庭以及合併使用厚朴酚和美滿庭都有顯著的神經保護效果。這些藥物在臨床上的使用值得作進一步的研究。
肆、研究雨傘節毒素(β-bungarotoxin)在腦部紋狀體注射的毒性反應,以及其與NMDA之交互作用
β-雨傘節毒素隸屬於IA型分泌性磷脂酶A2(type IA’ secretory phospholipaseA2)的一種,磷脂酶A2的作用是催化脂肪酸從磷脂的sn-2位置上裂解(cleavage)出來的化學反應。由於磷脂酶A2被認為與相當多的神經退化性疾病有關,因此研究磷脂酶A2在腦部缺血、阿茲海默症以及與興奮性神經毒性有關的神經傷害中扮演的角色,就顯得相當重要。在本實驗中,我們利用在鼷鼠的紋狀體內注射β-雨傘節毒素來研究分泌性磷脂酶A2在腦部造成的神經毒性,並進而研究其與NMDA的神經毒性有無差異性以及這兩者之間有無交互作用。我們將不同劑量的β-雨傘節毒素(1.5, 4.5, 7.5 μg/kg)、NMDA(0.15 mg/kg)以及NMDA(0.075 mg/kg)加上雨傘節毒素(0.75 μg/kg)注入鼷鼠(C57BL6)的紋狀體中,經過24小時後,我們將鼷鼠進行各種不同的神經行為測驗,如移動行為測驗、攀爬測驗、滾筒式跑步機平衡測驗、水迷宮(water maze)以及十字迷宮(plus maze)測驗等。隨後我們將鼷鼠犧牲,取出腦組織進行神經生化實驗(包括突觸體反應性氧化物、鈣離子的濃度以及粒線體還原酶活性等)。結果發現,在鼷鼠紋狀體注入β-雨傘節毒素會對上述的神經行為測驗,包括移動行為測驗、攀爬測驗、滾筒式跑步機平衡測驗、水迷宮以及十字迷宮測驗等,以及神經生化實驗包括突觸體反應性氧化物、鈣離子的濃度以及粒線體還原酶活性等產生與劑量相關(dose-dependent)的損害反應,;注入NMDA則無法測出具統計意義的顯著損害;而合併NMDA加上β-雨傘節毒素注射,在跳躍及攀爬測驗上,其損害則有加成(addition)的作用。因此,在β-雨傘節毒素所造成的腦部傷害,NMDA受體可能扮演的角色,尚待更進一步的研究。至於其他的穀氨酸鹽受體是否在β-雨傘節毒素所造成的腦部傷害扮演重要的角色,則必須有進一步的實驗來研究。
伍、結論:
在本論文中,我們藉由鼷鼠中大腦缺血的動物模式模擬腦中風,證明厚朴酚對鼷鼠中大腦缺血具有神經保護的作用。另外,我們利用在鼷鼠腦部的紋狀體注射NMDA造成興奮性神經毒性,並證明併用茶多酚及美滿庭在此動物模式具有比單用美滿庭更好的神經保護作用,此研究結果顯示並用美滿庭及茶多酚對一些臨床上由於興奮性神經毒性相關的疾病上,如腦部創傷、腦部缺血、癲癇、阿茲海默症等,具有應用的潛力。我們更進一步發展NMDA直接注入鼷鼠腦室的一種新的興奮性神經毒性動物模式,並利用這種動物模式證明單用厚朴酚、併用美滿庭與茶多酚或併用美滿庭與厚朴酚所具有神經保護的作用,均比單用美滿庭良。除此之外,我們利用β-雨傘節毒素單用或併用NMDA注入鼷鼠的紋狀體,比較研究β-雨傘節毒素以及NMDA的神經毒性以及其相互作用,結果證明β-雨傘節毒素會對神經行為測驗以及神經生化實驗產生與劑量相關的損害反應;注入NMDA則無法測出這樣的損害,但二者併用對跳躍及攀爬測驗有明顯的加成抑制作用。本實驗對我們後續將要繼續進行的神經保護藥物的研發上,也是一種相當重要的動物模式。總之,我們在這三種不同的動物腦部損害模式中,成功地證明NMDA受體拮抗劑以及抗氧化劑的併用,具有更好神經保護效果,奠定我們未來的研究,將以目前的成果作為基礎,希望能繼續研發出有效果而且能應用在臨床上的神經保護藥物,以期最終能達到治療這類疾病的目標。
With an incidence of approximately 250-400 in 100,000 and a mortality rate of around 30%, stroke remains the third leading cause of death in industrial countries. Brain tissue has a relatively high consumption of oxygen and glucose, and depends almost exclusively on oxidative phosphorylation for energy production. Focal impairment of cerebral blood flow restricts the delivery of substrates and impairs the energetics required to maintain ionic gradients. With energy depletion, membrane potential is lost and neurones and glia depolarize. Consequently, somatodendritic as well as presynaptic voltage-dependent Ca2+ channels become activated and excitatory amino acids are released into the extracellular space. Activation of NMDA receptors and metabotropic glutamate receptors contribute to Ca2+ overload. Activation of phospholipase A2 and cyclooxygenase generates free-radical species that overwhelm endogenous scavenging mechanisms, producing lipid peroxidation and membrane damage. In exploring the potential neuroprotective agent, we designed the following experiments:
1. Honokiol attenuates decreases in NA+, K+-ATPase activity and mitochondrial membrane potential and suppresses production of reactive oxygen species in the ischemic mouse brain
Honokiol, a component of the herb Magnolia officinalis, exhibits antioxidant, anti-inflammatory and anxiolytic properties, increases seizure threshold, and promotes neurite outgrowth. The present study was performed to ascertain whether honokiol also exerts neuroprotective effects in the ischemic brain. Adult male ICR mice were subjected to middle cerebral artery occlusion. Honokiol or saline were administered intraperitoneally 15 min before and 60 min after the induction of ischemia. Brain ischemia induced by this method was associated with an increase in synaptosomal production of reactive oxygen species, with decreases in synaptosomal mitochondrial membrane potential (△Ψm) and mitochondrial metabolic function, and with reductions in Na+, K+-ATPase activities of tissues isolated from selected brain regions. Administration of honokiol was followed by significant reductions in brain infarct volume and in synaptosomal production of reactive oxygen species. The decreases in synaptosomal mitochondrial membrane potential, synaptosomal mitochondrial metabolic function, and tissue Na+, K+-ATPase activities observed in the brains of these ischemic animals were also overturned by honokiol treatments. It is concluded that honokiol possesses the potential to protect the mammalian brain against ischemic-reperfusion injury and to preserve mitochondrial function during challenge by oxidative stress. Since ischemic stroke remains a leading cause of death in industrialized countries, further studies with honokiol are clearly warranted.
2. The novel regimen through combination of memantine and tea polyphenol for neuroprotection against brain excitotoxicity
NMDA receptors are abundant, ubiquitously distributed throughout the brain, fundamental to excitatory neurotransmission and critical for normal CNS function. However, excessive glutamate overstimulates NMDA receptors, leading to increased intracellular calcium and excitotoxicity. Mitochondrial dysfunction associated with the loss of Ca2+ homeostasis and enhanced cellular oxidative stress have long been recognized to play a major role in cell damage associated with excitotoxicity. In this experiment, we attempted to explore whether treatment with memantine (an NMDA receptor antagonist) and tea polyphenol (an antioxidant and anti-inflammatory agent), either alone or in combination, is effective in neuroprotection in a mouse excitotoxic injury model. Memantine (M, 10 mg/kg/day), tea polyphenol (TP, 60 mg/kg/day), or in combination (memantine 5 mg/kg/day plus tea polyphenol 30 mg/kg/day) were administered by oral gavage for 2 consecutive days before performing excitotoxic injury. Mice received 0.3 μl of NMDA (335 mM, pH, 7.2) injection into the left striatum. The locomotor activities were assessed 24 hours before and after excitotoxic injury. Brain synaptosomes were harvested 24 hours after excitotoxic injury for assessment of Na+, K+-ATPase and Mg2+ATPase activities, reactive oxygen species production, mitochondrial membrane potential (△Ψm), mitochondrial reductase activitiy (MTT test), and Ca2+ concentration. The results showed that treatment with memantine can significantly rescue mitochondrial function through attenuating the decreased mitochondrial membrane potential (△Ψm) and mitochondrial reductase activity in mouse excitotoxic injury. Treatment with tea polyphenol can significantly decrease the increased production of synaptosomal reactive oxygen species (ROS) and thus reduced the deteriorative ROS-sensitive Na+, K+-ATPase and Mg2+-ATPase activities. However, neither memantine nor tea polyphenol alone can significantly improve the impaired locomotor activities, unless treatment is combined. Combined treatment with memantine and tea polyphenol can significantly protect mice against excitotoxic injury by reducing the increased synaptosomal ROS production, attenuating all of the decreases in Na+, K+-ATPase and Mg2+-ATPase activities, mitochondrial membrane potential (△Ψm), mitochondrial reductase activity, and increased synaptosomal Ca2+ concentration. In addition, the impairment in locomotor activities was also significantly improved. Therefore, the combined treatment of memantine and tea polyphenol is more effective in neuroprotection than either memantine or tea polyphenol alone in mouse excitotoxic injury. These findings provide useful information with regards to the potential application of memantine and tea polyphenols for preventing clinical excitotoxic injury such as brain trauma, brain ischemia, epilepsy and Alzheimer’s disease.
3. Studies on excitotoxic animal modeling and the neuroprotective regimens of phytopolyphenols and memantine
Excitotoxicity is recognized to play a major role in various neurological disorders. In order to establish the effective therapeutic regimens toward excitotoxicity, we described a novel excitotoxic animal model in this study. By means of a direct injection of NMDA into cerebral ventricle of mice, we were able to elicit the various parameters of the neurotoxic effects, which allowed us to test the neuroprotective effects of various drug. Adult male ICR mouse was mounted on a stereotaxic frame, and 3 μl of NMDA (7 mM, pH, 7.2) was injected into the right ventricle (stereotaxic coordinates PA: –1.0 mm, lateral: +1.5 mm from bregma, and ventral: –2.5 mm relative to dura). Mice were treated with honokiol (1 mg/kg/day), memantine (10 mg/kg/day), tea polyphenol (60 mg/kg/day), either alone or in combination (honokiol 0.5 mg/kg or tea polyphenol 30 mg/kg/day plus memantine 5 mg/kg/day) for 2 consecutive days before performing excitotoxic injury. The occurrence of seizure activities of mice at the acute stage, followed by impairment in locomotor activity, climbing test, and motor equilibrium performance on rotarod 24 hours after injury were noted. Mice were sacrificed one day after NMDA administration, and the neurobiochemical parameters of the brain tissues were assayed. NMDA caused increases of synaptosomal ROS production and calcium concentration, decreases all of mitochondrial membrane potential, mitochondrial reductase activities, the neuronal membrane Na+, K+-ATPase, and Mg2+-ATPase activities. By comparing the score and severity of theses excitotoxic parameters, we were able to evaluate the neuroprotective regimens of various drug treatments. The results obtained revealed honokiol, tea polyphenol plus memantine,and honokiol plus memantine significantly attenuated the occurrence of generalized seizures (score 5 and 6), seizure score, the time latency of generalized seizures, the impairment in locomotor activity, climbing test, and motor equilibrium performance on rotarod. Moreover, the increases synaptosomal ROS production and intrasynaptosomal calcium concentration, or decreases in mitochondrial membrane potential, mitochondrial reductase activities, or membranous Na+, K+-ATPase, and Mg2+-ATPase activities were all significantly ameliorated. In conclusion, we have demonstrated a novel model of excitotoxicity by intraventricular administration of NMDA. Treatment with honokiol alone, or combined treatment with tea polyphenol, and memantine, and combined treatment with honokiol and memantine can ameliorate the impairments caused by excitotoxicity. Their therapeutic potential in treating excitoxicity-related diseases merits for further investigation.
4. Exploring the neurotoxic effects of intrastriatal injection of β-bungarotoxin and its interrelationship with NMDA
β-Bungarotoxin belongs to Type IA'' secretory phospholipase A2 toxin. PLA2 belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. Because PLA2s have been implicated in the pathology of a number of neurodegenerative diseases, an attempt was also made to elucidate the roles of PLA2s in cerebral ischemia, Alzheimer’s disease (AD), and neuronal injury due to excitotoxic agents. In this study, we utilize the intrastriatal injection of β-bungarotoxin to study the neurotoxic effects of sPLA2, and compare the different pathophysiological roles caused by β-bungarotoxin and NMDA in mouse brain to explore the interrelationship between these two agents. β-Bungarotoxin in different doses (1.5, 4.5, 7.5 μg/kg), NMDA (0.15 mg/kg mM), and NMDA (0.075 mg/kg) plus β-bungarotoxin (0.75 μg/kg) were administered in striatum (stereotaxic coordinates PA: –0.5 mm, lateral: -3.0 mm from bregma, and ventral: –4 mm relative to dura) of adult male C57BL6 mice (0.3 μL). Neurobehavior tests (locomotor activities, climbing test, rotarod, water maze, and plus maze) and neurobiochemical tests (DCF and fluo-3 fluorescence, MTT activity) were performed 24 hours after intrastriatal administration to explore their neurotoxic effects. The results showed administration of β-bungarotoxin in mouse striatum caused dose-dependent impairments in various neurobehavior tests and neurobiochemical tests. Administration of NMDA failed to induce such impairments. Further study is needed to clarify the role of other glutamate receptors in β-bungarotoxin-induced neurotoxicity.
5. Conclusion
In this doctoral thesis, we utilized the mouse MCAO model to mimic ischemic stroke, and proved that honokiol is neuroprotective in mouse MCAO model. Nextly, we developed mouse excitotoxicity model with intrastriatal injection of NMDA, and found that combined use of memantine and tea polyphenol is neuroprotective. These findings provide useful information with regards to the potential application of memantine and tea polyphenols for preventing clinical excitotoxic injury such as brain trauma, brain ischemia, epilepsy and Alzheimer’s disease. We further developed a novel excitotoxic model with intraventricular injection of NMDA, and found that treatment with honokiol alone, or combined treatment with tea polyphenol, and memantine, and combined treatment with honokiol and memantine can ameliorate the impairments caused by excitotoxicity. We also demonstrated that administration of β-bungarotoxin in mouse striatum caused dose-dependent impairments in various neurobehavior tests and neurobiochemical tests. Administration of NMDA failed to induce such impairments. These results provide important findings that we can explore potential neuroprotective agents in the future. In conclusion, we’ve developed several animal models of brain injury, and showed that combined use of NMDA receptor antagonist and antioxidant is neuroprotective. In the future, we’ll futher explore the potential neuroprotective agents that can be applied clinically. Further study will be done on the basis of these studies.
口試委員會審定書
誌謝………………………………………………………………………………… 3
中文摘要……………………………………………………………………………10
英文摘要………………………………………………………………………… 14
縮寫表…………………………………………………………………………… 23
PART 1: Background and introduction………………………………………… .24
1.1. Pathophysiology of ischemic stroke…………………………………………..24
1.2. Pathophysiology of excitotoxicity……………………………………………...26
1.3. Pharmacology of honokiol……………………………………………………..28
1.4. Pharmacology of tea polyphenol………………………………………………29
1.5. Animal models of excitotoxicity……………………………………………….30
1.6. Pathophysiology of β-bungarotoxin……………………………………...……31
1.7. Aims……………………………………………………………………………33
PART 2: Materials and methods…………………………………………………..34
2.1. Animal models of brain ischemia and excitotoxicity……………………………34
2.2. Drug administration……………………………………………………………..37
2.3. Experimental procedures………………………………………………………..38
2.3.1. Measurement of infarct volume………………………………………….……38
2.3.2. Seizure score…………………………………………………………………..39
2.3.3. Locomotor activity test………………………………………………………..40
2.3.4. Motor equilibrium performance on rotarod……………………………………40
2.3.5. Climbing test……………………………………………………………….…40
2.3.6. Water maze……………………………………………………………………41
2.3.7. Plus maze………………………………………………………………………41
2.3.8 Preparation of synaptosomes………………………………………………..….42
2.3.9. Measurements of Na+, K +-ATPase activity…………………………………...43
2.3.10. Measurement of reactive oxygen species formation………………………....44
2.3.11. Mitochondrial membrane potential……………………………………….….45
2.3.12. Assessment of mitochondrial metabolic function……………………………45
2.3.13. Measurement of intrasynaptosomal Ca2+ concentration……………………...46
2.3.14. Statistical analysis……………………………………………………………47
PART 3: Part 3: Results and discussion………………………………….……….48
I. Honokiol attenuates decreases in NA+, K+-ATPase activity and mitochondrial membrane potential and suppresses production of reactive oxygen species in the ischemic mouse brain……………………………………………………………….48
3.1.1. Results………………………………………………………………………...48
3.1.1.1 Reduction of brain infarct volume by honokiol……………………………...48
3.1.1.2. Reduction in brain Na+, K+-ATPase activity due to ischemia and attenuation of the reduction by honokiol………………………………………………………....48
3.1.1.3. Production of reactive oxygen species by synaptosomes of the ischemic brain and suppression of reactive oxygen species production by honokiol………………..49
3.1.1.4. Reduction of mitochondrial membrane potential (△Ψm) in synaptosomes from striatum and infarct areas of ischemic brain; attenuation of the reduction by honokiol………………………………………………………………………………51
3.1.1.5. Reduction of mitochondrial metabolic activity in synaptosomes from striatum and infarct areas of ischemic brain; attenuation of the reduction by honokiol………………………………………………………………………………52
3.1.2. Discussion……………………………………………………………………..53
II: The novel regimen through combination of memantine and tea polyphenol for neuroprotection against brain excitotoxicity……………….……………………..59
3.2.1. Results………………………………………………………………….……..59
3.2.1.1 Locomotor activities were impaired after excitotoxic injury, and attenuated by the combined treatment with memantine and tea polyphenol………………………..59
3.2.1.2. Na+, K+-ATPase and Mg2+-ATPaes activities decreased after excitotoxic injury, and preserved by treatment with tea polyphenol or combined treatment of memantine and tea polyphenol…………………………………………………………………...60
3.2.1.3. Increased production of reactive oxygen species after excitotoxic injury, and decreased by treatment with tea polyphenol or combined treatment with memantine and tea polyphenol……………………………………………………………….…..61
3.2.1.4. Mitochondrial membrane potential (△Ψm) decreased after excitotoxic injury, and preserved by treatment with memantine or combined treatment with memantine and tea polyphenol…………………………………………………………………...61
3.2.1.5. Mitochondrial reductase activity decreased after excitotoxic injury, and attenuated by treatment with memantine or combined treatment with memantine and tea polyphenol………………………………………………………………………..62
4.1.6. Intrasynaptosomal Ca2+ concentration increased after excitotoxic injury, and attenuated by treatment with memantine and tea polyphenol alone or in combination…………………………………………………………………………..62
3.2.2. Discussion……………………………………………………………….…….63
III: Studies on excitotoxic animal modeling and the neuroprotective regimens of phytopolyphenols and memantine………………………………….……………...69
3.3.1. Results……………………………………………………………………..….69
3.3.1.1. Time course of excitotoxic effects of various doses of NMDA………..……69
3.3.1.1.1. Early response………………………………………………….….………70
3.3.1.1.2 Late response………………………………………………….……………70
3.3.1.2. Neurobiochemical changes induced by NMDA…………………………….71
3.3.2. Discussion………………………………………………………………….….72
IV: Exploring the neurotoxic effects of intrastriatal injection of β-bungarotoxin and its interrelationship with NMDA…………………….………………………..80
3.4.1. Results………………………………………………...………………………80
3.4.2. Discussion……………………………………………....……………………..81
PART 4: Figures and tables……………………………..…….…………………..86
Figure 1 Chemical structures of honokiol, memantine, EGCG (tea polyphenol), NMDA, and β-bungarotoxin……………………………………………………..….86
Figure 2 Infarct areas of brains of ICR mice subjected to middle cerebral artery occlusion and treatment with saline or honokiol…………………………………….87
Figure 3 Na+, K+-ATPase activities of striatum, infarct areas, and non-infarct areas of brains of mice subjected to middle cerebral artery occlusion and treatment with saline or honokiol…………………………………………………………………………...88
Figure 4 Production of reactive oxygen species by synaptosomes after H2O2 challenge was attenuated by honokiol administration……………………………….89
Figure 5 Production of reactive oxygen species by synaptosomes prepared from striatum, infarct areas, and non-infarct areas of brains of mice subjected to middle cerebral artery occlusion and treatment with saline or honokiol……………………..90
Figure 6 Mitochondrial membrane potential (△Ψm) of synaptosomes from striatum, infarct areas, and non-infarct areas of brains of mice subjected to middle cerebral artery occlusion and treatment with saline or honokiol……………………………91
Figure 7 Mitochondrial metabolic activity of synaptosomes prepared from striatum, infarct areas, and non-infarct areas of brains of mice subjected to middle cerebral artery occlusion and treatment with saline or honokiol……………………….……..92
Figure 8 Locomotor activities of mice assayed before and after excitotoxic injury…………………………………………………………………………………93
Figure 9 Measurement of Na+, K+-ATPase, and Mg2+-ATPase activities in striatum and non-striatum areas of mice………………………………………………………94
Figure 10 Measurement of the production of synaptosomal reactive oxygen species in striatum and non-striatum areas of mice 24 hours after excitotoxic injury…………..95
Figure 11 Detection of mitochondrial membrane potential (△Ψm) in striatum and non-striatum areas of mice………………………………………………………...…96
Figure 12 Determination of mitochondrial reductase (MTT) activity in striatum and non-striatum areas of mice……………………………………………………..…….97
Figure 13 Determination of intrasynaptosomal Ca2+ concentration in striatum and non-striatum areas of mice 4 hours after excitotoxic injury…………………….……98
Figure 14 The changes of occurrence of generalized seizure, time latency (score 5 and 6) and their severity were recorded after drug treatment in mice………...…..….99
Figure 15 Locomotor activities of mice were tested before and after NMDA administration……………………………………..……………………………….100
Figure 16 Motor equilibrium performance on rotarod was tested before and after NMDA administration…………………………………………………..….………101
Figure 17 Changes of climbing score were recorded after NMDA administration……………………………………………………..…..……………102
Figure 18 Changes of ROS production, MMT, and mitochondria reductase activitiy of synaptosomal fractions were measured 24 hours after NMDA administration…….103
Figure 19 Changes of synaptosomal Na+, K+-ATPase and Mg2+-ATPase activities were measured 24 hours after NMDA administration……………………………...104
Figure 20 Changes of [Ca2+]i were measured 24 hours after NMDA administration. In N group, the fluorescence of fluo-3 was significantly increased……………...……105
Figure 21 Locomotor activities of mice were tested before and 24 hours after instrstriatal injection……………………………………………………………...…106
Figure 22 Climbing score were tested before and 24 hours after instrstriatal injection………………………………………………………………………..……107
Figure 23 Rotarod test was performed before and 24 hours after instrstriatal injection………………………………………………………………….………….108
Figure 24 Water maze test was performed before and 24 hours after instrstriatal injection……………………………………………………………………………..109
Figure 25 Plus maze test was performed before and 24 hours after instrstriatal injection……………………………………………………………………………..110
Figure 26 DCF fluorescence was measured 24 hours after instrstriatal injection…111
Figure 27 Fluo-3 fluorescence was measured 24 hours after instrstriatal injection..112
Figure 28 MTT activity was measured 24 hours after instrstriatal injection……...113
Table 1 Summary of drug effects on NMDA-induced excitotoxicity…..…………114
PART 5: Conclusion and future perspectives…………………………………..115
References…………………………………………………………………………118
List of publications………………………………………………………………..125
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