雖然在慢性疼痛的神經生物學機制已有密切的研究，但是慢性疼痛以目前藥物治療的效果仍是最令人不滿意的項目之一；麩胺酸鹽可激發兩種受器：離子性及metabotropic 受器，離子性受器又分為三類受器：AMPA、 kainate、 及NMDA 受器，脊椎及周邊組織中的NMDA受器之激發在動物實驗中，顯示在加強疼痛中佔有重要的角色，雖然NMDA受器拮抗劑在不同的實驗及臨床疼痛使用上均有效果，但是其中樞神經副作用也限制了其臨床上使用例如記憶力減低、精神狀態異常、運動失調等，目前有兩種可行的方式可獲得有效的止痛並避免中樞神經副作用：抑制中樞神經次類型的NMDA受器，或是在周邊使用麩胺酸鹽拮抗劑，如此不會影響中樞神經麩胺酸鹽運作，近來NMDA受器NR2B 被發現在脊髓中，而且局限在脊髓表層；近年來發現小段雙股RNA轉殖入動物細胞內，可導致對特定基因有效而長期的抑制，因此本實驗針對中樞及周邊神經之NMDA受器使用小段雙股RNA抑制基因方式及拮抗劑藥物止痛，第一種是椎管內注射NMDA受器NR2B小段雙股RNA並以polyethylenimine (PEI)將其轉殖入脊髓細胞內，福馬林用來引起老鼠後掌之發炎疼痛，在注射小段雙股RNA後之第3、7、14、21天觀察及記錄對福馬林之反應，脊髓在福馬林試驗之後馬上取出做mRNA及蛋白質之分析，結果顯示減少NR2B mRNA最高峰在椎管內注射5微克NR2B小段雙股RNA後第三天，蛋白質減少最高峰在第七天，NR2B mRNA及蛋白質減少持續十四天，並在第二十一天恢復，經由即時聚合酶連鎖反應(PCR)及西方墨點法測試出來，在第七天脊髓背角NR2B的組織免疫螢光染色也明顯減少，NR2B蛋白質減少的期間福馬林引起之疼痛也是減低，這些結果提供新的方法在脊椎內注射小段雙股RNA並以PEI轉殖入脊髓背角細胞可減低福馬林引起之疼痛；第二種是使用皮下注射NMDA受器拮抗劑或在組織表面給予alpha-2腎上腺受器激發劑以減少手術疼痛，此外我們也首次證實人類發炎皮膚的麩胺酸鹽受器是激增的，此實驗是將取自有發炎疼痛病人的發炎皮膚及附近區域皮膚作檢驗，使用即時聚合酶連鎖反應及西方墨點法分析正常及發炎皮膚離子性麩胺酸鹽受器mRNA及蛋白質量的改變，結果顯示在發炎狀態時麩胺酸鹽受器mRNA及蛋白質的表現是增加的，而這種麩胺酸鹽受器激增的現象也許和人類皮膚發炎疼痛傳導有關；為了測試皮下注射NMDA受器拮抗劑(ketamine)術後止痛作用及副作用，26位接受包皮手術的病人平均分成兩組分別接受皮膚切割前皮下注射3毫升0.3% ketamine 或saline，皮下注射saline的病人也接受上臂肌肉注射9毫克ketamine作為測試全身止痛作用的控制組，術後止痛作用及副作用觀察了24小時，皮下注射ketamine 的病人其術後之首次要求止痛劑的時間是延長的，同時活動及勃起時完全無痛的比率也明顯較控制組病人為高，另也無明顯的副作用，切割皮膚前於預定切割位置皮下注射ketamine經由周邊神經作用機制可抑制包皮手術術後疼痛；Apraclonidine hydrochloride (AH)是一種外用的腎上腺alpha-2受器激發劑，其不易進入中樞神經也產生較小副作用如昏睡及低血壓，80位接受膝關節鏡手術的病人分成四組，接受術後關節內注射saline、50微克AH、150微克AH、或150微克clonidine，結果關節內注射150微克AH、或150微克clonidine組，產生相似程度的術後止痛及相似的副作用，此研究提供了新的治療慢性疼痛並避免中樞神經副作用的兩大止痛方式。

Despite intensive research on the neurobiological mechanisms of chronic pain, this therapeutic area remains one of the least satisfactorily covered by current drugs. Glutamate activates two major classes of receptors: ionotropic and metabotropic. Ionotropic receptors are classified into three major subclasses:a-amino-3- hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). NMDA receptor activation, at the level of the spinal cord and peripheral tissue has been shown to play an important role in the facilitation of nociption in several animal models. Although the efficacy of NMDA receptor antagonists in various experimental and clinical pain situations has been well documented, their use as analgesics is limited by serious side effects such as memory impairment, psychotomimetic effects, ataxia and motor incoordination. Two promising current approaches to obtain effective analgesia devoid of side effects are by subtype-selective NMDA receptor antagonism in central nervous system (CNS) or peripheral use of NMDA receptor antagonist that do not interfere with central glutamate processing. NR2B subunit of NMDA receptor was predominantly found in the superficial dorsal horn of spinal cord. Recent discoveries have revealed that the transfection of small interfering RNAs (siRNAs) into animal cells results in the potent, long-lasting post-transcriptional silencing of specific genes. Thus, two approaches of antagonizing NMDA receptor in CNS and peripheral nervous system (PNS) for pain relief using siRNAs or pharmacological agents are investigated in this study. The first approach involves intrathecal administration of NR2B-siRNA into subarachnoid space and transfection of siRNA into cell of spinal cord by transfection agent of polyethylenimine (PEI). Formalin test was used to induce inflammatory pain in the hind paw of rats. Behavior response to formalin test was observed and recorded on 3rd, 7th, 14th, and 21th day after injection of siRNA. The spinal cords were dissected immediately after formalin test and used for analysis of mRNA and protein. The results revealed that the use of siRNA targeting the NR2B subunit could abolish formalin induced pain behaviors and not impair motor coordination in rat model. The expression of NR2B mRNA and its associated protein as demonstrated by real time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were decreased. Significant reduction of NR2B immunoreactivity in dorsal horn of spinal cord were detected after 7 days treated by NR2B siRNA. The peak effect of gene knockdown occurred on day 3 for mRNA and day 7 for its protein, following intrathecal injection of 5 µg of siRNA targeting NR2B subunit. The inhibition of NR2B mRNA and protein lasted about 14 days and recovered on 21th days after injection of siRNA. The nociceptive response induced by formalin was decreased during the period of downregulation of NR2B protein. A novel intrathecal delivery of siRNA transfected with PEI into cell of dorsal horn reduced formalin-induced pain. The second approach involves subcutaneous injection of NMDA receptor antagonist and topical use of alpha2-adrenergic agonist for abolishing surgical pain. Additionally, we proved the upregulation of glutamate receptors in human inflamed skin. The study examined whether the peripheral ionotropic glutamate receptors (iGluRs) increased in inflamed human skin taken from patients having inflammatory pain over inflamed skin and surrounding area. Real time RT-PCR and western blot were used for quantitation of mRNA and protein of iGluR in normal and inflamed human skin. A significant increase in mRNA and protein for the subunits of NMDA, AMPA, and kainate receptor were detected in inflamed skin when compared to normal skin. The results demonstrate that mRNA and protein level of iGluRs are increasingly expressed during states of persistent inflammation, and that this increased activity may be involved in mediating clinical inflammatory pain in human skin. To examine the postoperative analgesic effect and adverse effect of local NMDA receptor antagonist (ketamine), ketamine (0.3%, 3 ml) or saline was subcutaneous infiltrated pre-incisionally in 26 patients equally assigned to two groups undergoing circumcision surgery. The saline-infiltrated subjects also received 9-mg intramuscular ketamine into the upper arm to control for any related systemic analgesic effects. The postoperative analgesic and adverse effects were followed for 24 hours. For ketamine infiltrated patients, the time interval until first analgesic demand was prolonged and the incidence of pain free (pain score = 0) during movement and erection was significant higher than saline infiltrated patients. No significant differences were noted in the incidence of adverse effects between the two groups. Pre-incisional subcutaneous infiltration of ketamine acting via a peripheral mechanism can suppression postoperative pain after circumcision surgery. Apraclonidine hydrochloride (AH) is a topical, relatively selective alpha2-adrenergic agonist that has limited access to the CNS and exhibits fewer systemic (adverse) effects such as dizziness and hypotension. Eighty patients scheduled for arthroscopic knee surgery received either intraarticular (IA) normal saline, 50 ug IA AH, 150 ug IA AH, or 150 ug IA clonidine subsequent to surgery. The IA application of 150 ug apraclonidine and 150 ug clonidine provide similar degree of postoperative analgesia and similar incidence of adverse effect. The promise is that both approaches attenuating nociception state devoid of CNS adverse effects provide novel approach for the management of chronic pain.

論文口試委員審定書----------------------------------------i
致謝-----------------------------------------------------ii
摘要-----------------------------------------------------iii
Abstract-------------------------------------------------vi
1.0 Background--------------------------------------------1
1.1 Chronic Pain and Glutamate Receptor-------------------1
1.2 Subunit Composition and Functional Properties
of NMDA Receptor------------------------------------------2
1.3 NMDA Receptors in Central Nervous System--------------3
1.4 Glutamate Receptors in Peripheral Nervous System------5
1.5 Genetic and Pharmacological Pain Therapy in Central and Peripheral Nervous System--------------7
2.0 General Research Objectives--------------------------10
2.1 Specific Aims----------------------------------------10
3.0 Experimental Design----------------------------------13
3.1 Genetic Pain Therapy in Central Nervous System ------13
3.2 Pharmacological Pain Therapy in Peripheral Nervous System -------------------------14
4.0 Materials and Methods--------------------------------18
4.1 Synthesis of NR2B dsRNA -----------------------------18
4.2 Polymer Conjugate Synthesis--------------------------18
4.3 Behavioral Tests-------------------------------------18
4.4 RNA Isolation and Real-Time RT-PCR-------------------19
4.5 Western Blots----------------------------------------21
4.6 Immunohistochemistry of Spinal Cord Sections---------22
4.7 Anesthesia for Patients Receiving Circumcision Surgery -----------------------------23
4.8 Anesthesia for Patients Receiving Arthroscopic Surgery--------------------------23
4.9 Statistical Analysis --------------------------------24
5.0 Results----------------------------------------------26
5.1 Genetic Pain Therapy in Central Nervous System ------26
5.2 Pharmacological Pain Therapy in Peripheral Nervous System -----------------------28
6.0 Discussion-------------------------------------------31
7.0 References-------------------------------------------47
8.0 Table------------------------------------------------63
9.0 Figures and Legends----------------------------------71
Appendix-------------------------------------------------81
Publications and Presentations Related to This Thesis----88

Antonijevic I, Mousa SA, Schafer M, Stein C (1995) Perineurial defect and peripheral opioid analgesia in inflammation. J Neurosci 15:165-172.
Beirith A, Santos AR, Calixto JB (2002) Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Res 924:219-228.
Bernard JM, Hommeril JL, Passuti N, and Pinaud M (1991) Postoperative analgesia by iv clonidine. Anesthesiology 75:577-582.
Bolay H, Moskowitz MA (2002) Mechanisms of pain modulation in chronic syndromes. Neurology 59:S2-S7.
Bonnet F, Boico O, Rostaing S, Loriferne JF, and Saada M (1990) Clonidine-induced analgesia in postoperative patients: epidural versus intramuscular administration. Anesthesiology 72:423-427.
Boyce S, Wyatt A, Webb JK, O’Donnell R, Mason G, Rigby M, Sirinathsinghji D, Hill RG, Rupniak NM (1999) Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localization of NR2B subunit in dorsal horn. Neuropharmacology 38:611-623.
Brummelkamp T R, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553.
Butterworth JF and Strichartz GR (1993) The a2 – adrenergic agonists clonidine and guanfacine produce tonic and phasic block of conduction in rat sciatic nerve fibers. Anesth Analg 76:295-301.
Camarata PJ and Yaksh TL (1985) Characterization of the spinal adrenergic receptors mediating the spinal effects produced by microinjection of morphine into the periaqueductal gray. Brain Res 336:133-142.
Carlton SM, Chung K, Ding Z, Coggeshall RE (1998) Glutamate receptors on postganglionic sympathetic axons. Neurosci 83:601-605.
Carlton SM, Coggeshall RE (1999) Inflammation-induced changes in peripheral glutamate receptor populations. Brain Res 820:63-70.
Carlton SM, Hargett GL, Coggeshall RE (1995) Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin. Neurosci Lett 197:25-28.
Carlton SM, Zhou S, Coggeshall RE (1998) Evidence for the interaction of glutamate and NK1 receptors in the periphery. Brain Res 790:160-169.
Cavero I, Depoortere H, Lefevre-Borg F (1980) Pharmacological studies on para-aminoclonidine. Br J Pharmacol; 69:295-296.
Chen L, Huang LY (1992) Protein kinase C reduces Mg2+block of NMDA-receptor
channels as a mechanism of modulation. Nature 356:521-523.
Coderre TJ, Basbaum AI, Helms C, Levine JD (1991) High dose epinephrine acts at alpha 2-adrenoreceptors to suppress experimental arthritis. Brain Res 544: 325-328.
Coderre TJ, Melzack R (1992) The contribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury. J Neurosci 12:3665-3670.
Coderre TJ, Vaccarino AL, Melzack R (1990) Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 535:155-158.
Coggeshall RE, Carlton SM (1998) Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery. J Comp Neurol. 391:78-86.
Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37:205-237.
Cook AJ, Woolf CJ, Wall PD, McMahon SB (1987) Dynamic receptive field plasticity in rat spinal cord dorsal horn following C-primary afferent input. Nature 325:151-153.
Crawley JN, Paylor R (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Behav 31:197-211.
Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42-51.
Cull-Candy S, Brickley S, Farrant M (2001) NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11:327-335.
Davidson EM, Coggeshall RE, Carlton SM (1997) Peripheral NMDA and non-NMDA glutamate receptors contribute to nociceptive behaviors in the rat formalin test. Neuroreport 8:941-946.
deGroot J, Zhou S, Carlton SM (2000) Peripheral glutamate release in the hindpaw following low and high intensity sciatic stimulation. Neuroreport 11:497-502.
Demeneix B, Behr J, Boussif O, Zanta MA, Abdallah B, Remy J (1998) Gene transfer with lipospermines and polyethyleneimines. Adv Drug Deliv Rev 30:85-95.
Dickenson AH, Sullivan AF (1987) Evidence for a role of the NMDA receptor in frequency dependent potentiation of deep rat dorsal horn nociceptive neurons following C fibre stimulation. Neuropharmacology 26:1235-1238.
Dougherty PM, Staats PS (1999) Intrathecal drug therapy for chronic pain. Anesthesiology 91:1891-1918.
Doubell TP, Mannion RJ, Woolf CJ (1999) The dorsal horn: state dependent
sensory processing, plasticity and the generation of pain. In: Wall PD, Melzack R, eds. Textbook of pain. London: Churchill Livingstone, 165-181.
Dowdy EG, Kaya K, Gocho Y (1973) Some pharmacologic similarities of ketamine, lidocaine, and procaine. Anesth Analg 52:839-842.
Drummond PD (1995) Noradrenaline increases hyperalgesia to heat in skin sensitized by capsaicin. Pain 60:311-315.
Du J, Koltzenburg M, Carlton SM (2001) Glutamate induced excitation and sensitization of nociceptors in rat glabrous skin. Pain 89:187-198.
Dubuisson D, Dennis SG (1977) The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4:161-174.
Durrani Z, Winnie AP, Zsigmond EK, Burnett ML (1989) Ketamine for intravenous regional anesthesia. Anesth Analg 68:328-332.
Eisenach JC, Detweiler D, Hood D (1993) Hemodynamic and analgesic actions of epidurally administered clonidine. Anesthesiology 78: 277-287.
Eisenach JC, Lavand’homme P, Tong C, Cheng JK, Pan HL, Virtanen R, Nikkanen H, James R (1999) Antinociceptive and hemodynamic effects of a novel