氯胺酮是一種麻醉劑，也是一種濫用藥物。近年來，氯胺酮濫用增長迅速，在香港已經成爲第二大濫用藥物。氯胺酮的短期效果主要導致精神狀態改變，但對其長期效果還了解甚少。研究目的：本研究旨在探討長期使用氯胺酮對中樞神經系統，腎上腺，胰腺和膀胱的影響。研究方法：我們在氯胺酮濫用的動物模型中進行的行爲學，神經化學，組織學和分子生物學研究。在水迷宮中，對這些小鼠學習和記憶能力進行了評估。應用基因芯片評估中樞神經系統的基因表達變化。用聚合酶鏈式反應陣列研究神經遞質及其調控基因表達變化。通過實時定量聚合酶鏈式反應和免疫印迹法檢測-氨基丁酸受體和多巴胺相關基因的基因表達的變化。用酶聯免疫法測定多巴胺含量。用原位末端轉移酶標記技術染色（細胞凋亡），天狼星紅染色（纖維化），免疫組織化學（乳酸脫氫酶，酪氨酸羟化酶，多巴胺β羟化酶）研究腎上腺，胰腺，膀胱癌的組織學變化。研究結果：與對照組相比，氯胺酮組小鼠學習記憶能力下降。基因芯片結果顯示，110個基因表達上調和136個基因表達下調。基因本體分析表明，氯胺酮明顯影響神經遞質和受體的活性。特別地，4-氨基丁酸A受體5型亞基的mRNA和蛋白水平在前額皮層的顯著上調。聚合酶鏈式反應陣列結果表明，氯胺酮顯著改變-氨基丁酸受體，神經肽，多巴胺和膽鹼能系統基因表達。對多巴胺系統的研究結果顯示，中腦多巴胺含量上調，酪氨酸羟化酶的顯著增加。在腎上腺和胰腺，氯胺酮和氯胺酮加酒精組都未觀察到細胞凋亡增加，但是觀察到乳酸脫氫酶的陽性染色。此外，在腎上腺中發現酪氨酸羟化酶和多巴胺β羟化酶下調。在膀胱中，在肌肉層觀察到細胞凋亡和纖維化。結論：本研究的結果研究指出，長期使用氯胺酮能引起中樞神經系統異常的基因表達，還能導致腎上腺，胰腺，膀胱癌的病理性改變。這些結果爲氯胺酮濫用相關的健康風險評估提供了重要的信息。 / Ketamine is an anesthetic agent and a drug of abuse. In recent years, ketamine abuse has been increasing rapidly and it has become the second-most popular abusive drug in Hong Kong. While the acute effects of ketamine are mainly linked to altered mental status, the long-term effects of ketamine are poorly understood. Objectives: The present study was designed to investigate the long-term effects of ketamine on the CNS, adrenal, pancreas and urinary bladder. Methods: Behavioral, neurochemical, histological and molecular studies were performed in a ketamine abuse animal model. Learning and memory ability in these mice were assessed in a morris water maze. An Affymetrix Genechip study was performed to assess the global gene expression changes in the CNS and a PCR-array study focused on the neurotransmitters and regulators was also performed. Gene expression changes for gamma-aminobutyric acid (GABA) receptors and dopamine related genes were assay by real-time PCR and western blot. Dopamine contents were measured by ELISA. Histological changes in adrenal, pancreas and urinary bladder were examined by TUNEL staining (apoptosis), Sirius red staining (fibrosis), and immunohistochemistry. Results: Compared with saline controls, there was a decline in learning and memory performance in the ketamine-treated mice. Genechip results showed that 110 genes were up-regulated and 136 genes were down-regulated in ketamine group. An ontology analysis revealed the most significant effects of ketamine were on neurotransmitter and receptor activities. In particular, there was a significant up-regulation of both mRNA and protein levels of the alpha 5 subunit (Gabra5) of the GABAA receptors in the prefrontal cortex. Results from the PCR-array study revealed significant gene expression changes in the GABA receptors, neuropeptides, dopaminergic and cholinergic system following ketamine treatment. Studies on the DA system revealed significant increase of DA content and up-regulation of Tyrosine Hydroxylase (TH) in the midbrain. In the adrenal and pancreas, no obvious apoptosis was found while lactate dehydrogenase (LDH) positive staining was observed in both ketamine and ketamine plus alcohol treated groups. On top of these, downregulation of TH and DBH were observed. In the urinary bladder, apoptosis and fibrosis were observed in the muscular layer. Conclusion: The present study pointed out that long-term of ketamine use caused aberrant gene expression in the CNS and led to pathological changes in adrenal, pancreas and urinary bladder. These results have provided novel and important insights in evaluating the health risks in ketamine abusers. / Detailed summary in vernacular field only. / Tan, Sijie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 138-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘 要 --- p.III / List of abbreviations --- p.IV / Acknowledgements --- p.VI / Contents --- p.VII / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Ketamine and abuse --- p.1 / Chapter 1.2 --- Pharmacological effects of ketamine --- p.4 / Chapter 1.3 --- Effects of ketamine on the CNS --- p.6 / Chapter 1.4 --- GABA receptors --- p.9 / Chapter 1.5 --- Dopamine system in the CNS --- p.10 / Chapter 1.6 --- The modulation of dopaminergic neurons --- p.12 / Chapter 1.7 --- Toxic effects of ketamine on other organs --- p.14 / Chapter 1.8 --- Thesis outline --- p.17 / Chapter Chapter 2 --- Cognition and GABA receptor expression following long-term ketamine administration --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.22 / Chapter 2.2.1 --- Animals and drug administrations --- p.22 / Chapter 2.2.3 --- Dosage Determination --- p.23 / Chapter 2.2.4 --- Morris water maze --- p.24 / Chapter 2.2.5 --- Brain tissue collection and RNA extraction --- p.25 / Chapter 2.2.6 --- Microarray analysis --- p.26 / Chapter 2.2.7 --- Quantitative real-time PCR --- p.27 / Chapter 2.2.8 --- Western blotting --- p.28 / Chapter 2.2.9 --- Statistical analysis --- p.29 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- Morris water maze --- p.29 / Chapter 2.3.2 --- Microarray analysis --- p.30 / Chapter 2.3.3 --- Quantitative real-time PCR --- p.30 / Chapter 2.3.4 --- Western blotting --- p.31 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- PCR-array gene expression profiling on the neurotransmitters following chronic ketamine administration --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and methods --- p.50 / Chapter 3.3 --- Results --- p.51 / Chapter 3.4 --- Discussion --- p.63 / Chapter Chapter --- 4 Chronic ketamine administration modulates midbrain dopamine system in mice --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.69 / Chapter 4.2.1 --- Cell culture and ketamine treatment --- p.69 / Chapter 4.2.2 --- MTT assay --- p.70 / Chapter 4.2.3 --- Animals and ketamine administration --- p.70 / Chapter 4.2.4 --- Dopamine determination --- p.71 / Chapter 4.2.5 --- Real-time PCR --- p.72 / Chapter 4.2.6 --- Western blotting --- p.73 / Chapter 4.2.7 --- Immunohistchemistry --- p.74 / Chapter 4.2.8 --- Statistical analysis --- p.74 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of ketamine on dopamine concentrations in PC12 cells --- p.75 / Chapter 4.3.2 --- Long-term effects of ketamine on dopamine in mouse brain --- p.76 / Chapter 4.3.3 --- Effects of ketamine on mRNA levels of dopamine related genes --- p.76 / Chapter 4.3.4 --- Long-term effects of ketamine on BDNF protein levels in mouse brain --- p.77 / Chapter 4.3.5 --- Increased TH inmmureactive neurons in midbrain following 3 months ketamine treatment --- p.78 / Chapter 4.4 --- Discussion --- p.90 / Chapter Chapter 5 --- Chronic treatment of ketamine affects adrenal gland and pancreas --- p.95 / Chapter 5.1 --- Introduction --- p.95 / Chapter 5.2 --- Materials and methods --- p.95 / Chapter 5.2.1 --- Grouping of experimental animals and treatments --- p.95 / Chapter 5.2.2 --- Histological studies on pancreas and adrenal --- p.96 / Chapter 5.2.3 --- Immunohistochemistry on pancreas and adrenal --- p.97 / Chapter 5.2.4 --- TUNEL evaluation --- p.99 / Chapter 5.3 --- Results --- p.100 / Chapter 5.4 --- Discussion --- p.114 / Chapter Chapter 6 --- Ketamine Effects on the Urogenital System-Changes in the Urinary Bladder and Sperm Motility --- p.117 / Chapter 6.1 --- Introduction --- p.117 / Chapter 6.2 --- Materials and methods --- p.117 / Chapter 6.2.1 --- Studies of the Bladder --- p.117 / Chapter 6.2.2 --- Studies on Sperm Motility --- p.120 / Chapter 6.3 --- Results --- p.121 / Chapter 6.4 --- Discussion --- p.131 / Chapter Chapter 7 --- Conclusion --- p.134 / Chapter 7.1 --- Conclusion --- p.134 / Chapter 7.2 --- Future studies --- p.137 / Bibliography --- p.138

本研究的目的是評估長期服用氯胺酮對青少年認知功能和精神健康狀況的影響。 自2009年12月至2011年12月，共300名受試者入組。受試者分為3組：氯胺酮組，氯胺酮及多種藥物組和健康對照組，每組有100名受試者入組。精神狀況評估包括問卷篩查和麵談。所有受試者均完成一套詳細的認知測試。該測試涵蓋一般智慧、語詞記憶、視覺記憶、執行功能、動作速度和語言。 / 氯胺酮組受試者主要濫用氯胺酮，而氯胺酮及多種藥物組受試者除氯胺酮外主要濫用可卡因和冰毒。兩組氯胺酮濫用者最常見的共患精神障礙是抑鬱障礙。在單因素分析中,兩組氯胺酮濫用者在幾乎所有的測試中得分低於健康對照。多因素分析控制混雜因素如年齡、性別、教育程度和Beck 抑鬱量表總分後，兩組氯胺酮濫用組與健康對照組在語詞記憶和視覺記憶仍存在顯著差異。本研究進一步將氯胺酮組及氯胺酮多種藥物組分別分為現用藥者和戒斷者。在氯胺酮組中現用藥者在詞記憶、視覺記憶、動作速度和部分執行功能測試上得分低於戒斷者和健康對照，並且現用者Beck 抑鬱量表總分高於戒斷者和健康對照，而戒斷者和健康對照在認知測試和Beck 抑鬱量表總分沒有顯著差別。但在氯胺酮及多種藥物組，現用藥者和戒斷者均在記憶測試中得分低於及Beck 抑鬱量表總分高於健康對照。另外, 女性氯胺酮濫用組視覺記憶得分低於男性，但女性在語詞記憶得分普遍高於男性。 / 本研究認為氯胺酮或氯胺酮合用多種藥物均能導致記憶和執行功能的損害。這種損害主要與近期濫用氯胺酮有關，並且氯胺酮組現用藥者語詞記憶損害較氯胺酮合用多種藥物現用藥者嚴重。單純氯胺酮導致的記憶和執行功能損害在戒斷1月後明顯好轉但氯胺酮合用多種藥物者戒斷一月後未能見到記憶功能好轉。超過半數的氯胺酮濫用者共患抑鬱障礙。本研究的結果為治療氯胺酮濫用有用資訊，亦有助於戒毒者鞏固其戒斷行為。但氯胺酮所致認知損害的可逆性還需要前瞻性或縱向研究進一步證實，並且這種可逆性損害的機制還不明確。未來的研究還需要進一步明確氯胺酮對人體的損害作用是否具有性別差異性。 / The objective of this study was to evaluate the long-term effect of ketamine use on both the cognition and psychological well-being of youths in Hong Kong. / Three hundred participants were recruited for the study, which lasted from December 2009 to December 2011. Participants were divided into three groups of 100 each: primarily ketamine (Primarily K) users, poly-drug ketamine (Poly K) users and healthy controls (HCs). Psychiatric assessments included screening with self-rating questionnaires and face-to-face interviews. All participants completed a detailed cognitive battery covering general intelligence, verbal memory, visual memory, executive function, motor speed and language. / The participants in the Primarily K group predominantly used ketamine, whereas those in the Poly K group used ketamine in addition to secondary drugs, of which cocaine and methamphetamine were the most frequent. Depressive disorder was the most common psychiatric disorder in both ketamine groups. Univariate analysis also showed the two ketamine groups to score poorly on most of the cognitive tests relative to the HC group. After adjusting for age, sex, education and Beck Depression Inventory (BDI) score, verbal and visual memory remained impaired in both ketamine groups in comparison with the HC group. Ketamine use in the past month was independently related to memory impairment in the Primarily K group. In subgroup analyses of Primarily K users, verbal and visual memory, motor speed, and some of the executive function indexes were significantly impaired in current users but not in ex-users. These findings suggest that the cognitive influence of ketamine is reversible. Moreover, the current ketamine users had a higher BDI score than the ex-users or HCs. However, the ex- and current poly-drug ketamine users exhibited a similar degree of memory impairment compared with the HCs. The female Primarily K users showed more visual memory impairment than their male counterparts, although females generally performed better than males in verbal memory. / In conclusion, the use of ketamine alone and in conjunction with other psychotropic drugs is associated with deficits in memory and executive function. The observed memory impairment was related primarily to recent ketamine use, with current Primarily K users presenting with a more severe memory deficit than current Poly K users. However, the Primarily K group realised improvement in cognitive impairment after abstaining from ketamine, whereas the Poly K group did not. In addition to cognitive functioning difficulties, more than half of the ketamine users suffered from depressive disorder. Moreover, the findings suggest that women may be more sensitive than men to visual memory impairment following chronic ketamine use. The findings of this study will be helpful in treating ketamine abuse, and reinforce the efficacy of abstinence from drugs. Further longitudinal research is needed to determine the reversibility of ketamine’s effects and the mechanism by which that reversibility takes place. Further study is also needed to clarify the drug’s sex-specific effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Huajun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 102-119). / Abstract also in Chinese. / DECLARATION OF ORIGINALITY --- p.I / ACKNOWLEDGEMENTS --- p.II / PUBLICATIONS AND PRESENTATIONS --- p.1 / LIST OF ABBREVIATIONS --- p.XI / LIST OF TABLES AND FIGURES --- p.XIII / ABSTRACT --- p.XV / 摘要 --- p.XVIII / CONTENTS --- p.XX / Chapter CHAPTER 1 --- BACKGROUND --- p.1 / Chapter 1.1 --- Introduction to ketamine and ketamine misuse --- p.1 / Chapter 1.2 --- Effects profile of ketamine in the brain --- p.4 / Chapter 1.2.1 --- Glutamate system dysfunction underlying ketamine actions --- p.4 / Chapter 1.2.2 --- Different effect patterns between acute and repeated administration --- p.7 / Chapter 1.2.3 --- Acute and chronic effects of ketamine on human cognitive functions --- p.9 / Chapter 1.2.4 --- Sex differences in addiction --- p.11 / Chapter 1.3 --- Introduction to cognition and intelligence and their assessments --- p.13 / Chapter 1.3.1 --- Memory and memory assessments --- p.13 / Chapter 1.3.2 --- Executive function and assessments of executive function --- p.16 / Chapter 1.3.3 --- Intelligence and intelligence tests --- p.19 / Chapter 1.4 --- Study hypotheses --- p.31 / Chapter CHAPTER 2 --- METHODS --- p.29 / Chapter 2.1 --- Study design --- p.29 / Chapter 2.2 --- Study subjects --- p.31 / Chapter 2.2.1 --- Subject recruitment sites --- p.31 / Chapter 2.2.2 --- Inclusion criteria --- p.32 / Chapter 2.3 --- Data collection --- p.33 / Chapter 2.3.1 --- Demographic information --- p.33 / Chapter 2.3.2 --- Drug use pattern and severity --- p.34 / Chapter 2.3.3 --- Psychiatric comorbidities --- p.34 / Chapter 2.3.4 --- Cognitive function evaluation --- p.40 / Chapter 2.4 --- Statistical methods --- p.44 / Chapter CHAPTER 3 --- RESULTS --- p.45 / Chapter 3.1 --- Demographics and basic information --- p.45 / Chapter 3.2 --- Drug use patterns --- p.47 / Chapter 3.3 --- Comorbid psychiatric problems --- p.51 / Chapter 3.4 --- Cognitive functions --- p.59 / Chapter 3.4.1 --- Cognitive functions among primarily ketamine, poly-drug ketamine and control groups --- p.59 / Chapter 3.4.2 --- Cognitive functions in current and ex-primarily ketamine users --- p.60 / Chapter 3.4.3 --- Cognitive functions in current and ex-poly-drug ketamine users --- p.70 / Chapter 3.4.4 --- Cognitive functions in current primarily ketamine and current poly-drug ketamine users --- p.79 / Chapter 3.4.5 --- Cognitive functions in female and male primarily ketamine users --- p.80 / Chapter CHAPTER 4 --- DISCUSSION --- p.86 / Chapter 4.1 --- Demographics and drug use patterns --- p.86 / Chapter 4.2 --- Effects of ketamine on psychological health --- p.87 / Chapter 4.3 --- Effects of ketamine on cognitive functions --- p.88 / Chapter 4.3.1 --- Effects of primarily ketamine use on memory --- p.90 / Chapter 4.3.2 --- Effects of primarily ketamine use on executive functions --- p.92 / Chapter 4.3.3 --- Effects of poly-drug ketamine use on cognitive functions --- p.96 / Chapter 4.3.4 --- Sex-specific effects of ketamine use on cognitive functions --- p.98 / Chapter CHAPTER 5 --- LIMITATIONS AND CONCLUSIONS --- p.98 / Chapter 5.1 --- Limitations --- p.98 / Chapter 5.2 --- Conclusions --- p.99 / REFERENCES --- p.102

Made available in DSpace on 2014-12-17T14:16:34Z (GMT). No. of bitstreams: 1 AnaLSAM_DISSERT.pdf: 1550415 bytes, checksum: caf1d01895bb5be79ca908c1ce3bf0f9 (MD5) Previous issue date: 2013-03-19 / Panax ginseng CA Meyer (Araliaceae) is a herbaceous plant widely used in China, South Korea, Japan and other Asian countries for the treatment of various diseases micro circulatory, cerebrovascular, among others, representing one of the drugs used by older man. It has over 30 biologically active ginsenosides with different pharmacological and behavioral effects and inhibitory effect on the NMDA receptor. The amino acid glycine is a co-agonist of the NMDA receptor, activating this receptor. At the cellular level, ketamine is widely known to be NMDA receptor antagonist. The aim of this study was to evaluate the general activity in the open field, and anxiety in elevated plus maze, mice treated with P. ginseng compared with the action of ketamine and glycine, to better understand the action of this herbal medicine at the NMDA receptor. We used 66 adult male rats were divided into six groups: a positive control, treated for 30 days with water by gavage, who received glycine (500mg/kg; po) on days 7, 14, 21 and 28 of treatment, one hour before of behavioral assessment, a negative control was treated for 30 days with water by gavage received ketamine (5mg/kg, ip) on days 7, 14, 21 and 28 of treatment, one hour prior to behavioral evaluation, three experimental groups, receiving 100, 200 or 300 mg / kg P. ginseng by gavage for 30 days and one group treated solely with white water, and is also administered 1 ml of water by gavage one hour prior to behavioral evaluation. Animal behavior in these three groups was also examined on days 7, 14, 21 and 28 of treatment. On day 30 of treatment, the animals were anesthetized with thiopental (70mg/kg) for blood collection and after euthanasia, withdrawal of various organs. There were no changes in weight and body weight gain and weight reasons in organ / body weight. However the consumption of water and food values showed a significant increase. Serum levels of AST was increased in a dose-dependently in the animals treated with doses of P. ginseng, glycine and ketamine as compared to the blank group. Unlike creatinine levels proved to be decreased in all treated groups when compared with white. However, the level of urea in these groups was reduced and no changes were observed in the ALT parameter. Histopathological examination revealed no changes in cell morphology in different tissues. There were no behavioral changes in the elevated plus maze and few changes were observed in the open field, animals treated with P. ginseng, glycine and ketamine when compared to white. These data suggest that the doses of P. ginseng employed were unable to induce general toxicity in rats treated for 30 days and also shows that the general behavior of mice treated with P. ginseng was slightly different from that observed in animals treated with ketamine and glycine. Finally, the study on the elevated plus maze showed that the extract of P. ginseng showed no anxiolytic or anxiogenic action / Panax ginseng C.A. Meyer (Araliaceae) ? uma planta herb?cea muito usada na China, Cor?ia do Sul, Jap?o e outros pa?ses da ?sia no tratamento de v?rias doen?as micro circulat?rias, vasculares cerebrais, entre outras. Possui mais de 30 ginsenos?deos, que inibem o receptor NMDA, provocando diferentes efeitos farmacol?gicos e comportamentais. O objetivo do presente estudo foi avaliar a atividade geral, no campo aberto, e a ansiedade, no labirinto em cruz elevado, de ratos tratados com P. ginseng. Ratos tratados com ketamina (antagonista do receptor NMDA) e com glicina (coagonista do receptor NMDA), foram tamb?m empregados para melhor entendimento do mecanismo de a??o desse fitoter?pico. Foram utilizados 66 ratos machos adultos, divididos em seis grupos: um controle positivo (n=12), tratado durante 30 dias com ?gua por gavagem, que recebeu glicina (500mg/kg; v.o.) nos dias 7, 14, 21 e 28 de tratamento, uma hora antes da avalia??o comportamental; um controle negativo (n=12), tratado durante 30 dias com ?gua por gavagem, que recebeu ketamina (5mg/kg; i.p.) nos dias 7, 14, 21 e 28 de tratamento, uma hora antes da avalia??o comportamental; tr?s grupos experimentais (n=12), que receberam 100, 200 ou 300 mg/kg de P. ginseng, por gavagem, durante 30 dias e um grupo branco (n=6) tratado exclusivamente com ?gua, sendo tamb?m administrado 1mL de ?gua por gavagem uma hora antes da avalia??o comportamental. O comportamento animal nesses grupos tamb?m foi analisado nos dias 7, 14, 21 e 28 de tratamento. No dia 30 de tratamento os animais foram anestesiados para coleta de sangue e retirada de ?rg?os diversos, que tiveram seus pesos anotados e por??es foram coletadas para estudo histopatol?gico. N?o foram observadas altera??es no peso e ganho de peso corporal entre os diversos grupos nem nas raz?es peso ?rg?o/peso corporal calculadas. Nos animais tratados com P. ginseng, ketamina e glicina o consumo de ?gua e de ra??o e as concentra??es s?ricas de AST revelaram estar aumentadas em compara??o com grupo branco. Entretanto, os animais tratados com as tr?s doses de P. ginseng, ketamina e glicina apresentaram n?veis reduzidos de creatinina e ureia quando comparados com o grupo branco. N?o foram observadas altera??es no par?metro ALT. O estudo histopatol?gico revelou aus?ncia de altera??es na morfologia celular nos diversos tecidos analisados. N?o foram encontradas altera??es comportamentais no labirinto em cruz elevado e poucas altera??es foram observadas nos animais tratados com P. ginseng, glicina e ketamina quando comparados com o grupo branco, no campo aberto. Esses dados sugerem que as doses de P. ginseng empregadas n?o foram capazes de provocar toxicidade geral em ratos tratados por 30 dias e revela tamb?m que o comportamento geral dos ratos tratados com P. ginseng foi pouco diferente daquele observado nos animais tratados com glicina e ketamina. Por fim, o estudo no labirinto em cruz elevado mostrou que o extrato de P. ginseng n?o apresentou a??o ansiog?nica nem ansiol?tica nas condi??es experimentais adotadas