以大鼠大腦微透析模式探討尼古丁對MPTP/TIQ通過血腦屏障(BBB)影響之研究

Effects of nicotine on Blood-Brain Barrier (BBB) transfer of MPTP/ TIQ-an in vivo brain microdialysis study

張祐嘉(Yu-Chia Chang)

指導教授 ： 林君榮

國立臺灣大學/醫學院/藥學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.03198

摘要

巴金森氏症是一種與年齡有關係的黑質紋狀體神經退化疾病，它會導致病人產生行動遲緩、靜止型顫抖、姿勢障礙和肌肉僵硬。一種具有選擇性在多巴胺神經產生毒性的物質MPTP，在給予之後會造成類似巴金森氏症的症狀。往後，MPTP就被用來建立各種適合的動物模式來進一步研究巴金森氏症相關的神經死亡以及神經保護作用。
流行病學的研究顯示出吸煙者與罹患巴金森氏症具有負相關性。為了探討香煙中主要成分尼古丁對於血腦障壁運送引起巴金森氏症的物質的影響，我們使用大腦微透析技術收集大鼠腦紋狀體的透析液進行研究。先將雄性Wistar大鼠分成2大組。一組腹腔注射生理食鹽水，另一組則是在注射MPTP前30分鐘給予單一劑量的尼古丁(0.3, 0.6, 2.0 and 4.0 mg/ kg)。然後在腹腔注射生理食鹽水或是尼古丁30分鐘後由股靜脈給予MPTP (10 mg/ kg)輸注。透析液以輸注MPTP後開始收集，每30分鐘一管共收取150分鐘。MPTP與MPP+的濃度以高效能液相層析系統進行定量，此系統使用C18層析管柱以及紫外光與螢光光譜偵測儀。結果顯示，在尼古丁的作用下，第0~30分鐘腦細胞外液中的MPTP量有明顯地減少(666.31±65.53 ng/ mL, 561.50±12.11 ng/ mL, 493.23±35.46 ng/ mL, 375.94±76.24 ng/ mL 以及 329.35±55.14 ng/ mL分別代表控制組與用0.3, 0.6, 2.0, 4.0 mg/ kg尼古丁前給予的組別)。此時我們也監測給予0.6 mg/ kg尼古丁的腦中以及血中濃度，結果顯示尼古丁在第30~60分鐘(相當於收集MPTP的第0~30分鐘)是濃度達到最高的時段(136.8±16.6 ng/mL, 233.1±9.2 ng/mL)。另外我們以上述建立的系統，也進一步探討投予次數與性別是否會影響尼古丁的作用。結果顯示，在多次劑量尼古丁的給予下，MPTP量的確有比控制組少，但是其改變量與單一劑量尼古丁比較，並沒有因投予尼古丁次數增加而有明顯地減少。在性別上，控制組與各劑量尼古丁的MPTP量，有明顯減少，但是尼古丁的作用並沒有因性別差異而在效力上有明顯的不同。另外，Tetrahydroisoquinoline (TIQ)是一個與MPTP結構類似且會引起巴金森氏症的物質。我們同樣也研究了尼古丁對於血腦障壁運送TIQ的影響。結果顯示，腦細胞外液中的TIQ並沒有因為事先給予尼古丁(0.6, 2.0 mg/kg)而有明顯減少。
總結本實驗的結果，我們發現尼古丁確實可以減少MPTP被運送過血腦障壁至腦中，但是與MPTP結構相似的TIQ，尼古丁無法有效減少它被運送過血腦障壁。有關為何抽煙有較低巴金森氏症發生率的詳細機轉可能還需要進一步確認。

關鍵字

微透析 ； 尼古丁 ； 血腦屏障

並列摘要

Parkinson’s disease (PD) is an age-related neurodegeneration disorder resulting in bradykinesia, resting tremor, postural instability and muscle rigidity. Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective dopaminergic neurotoxin, causes a Parkinson-like syndrome. In this regard, MPTP is used to establish suitable animal models to study neurodegenerative and neuroprotective processes in PD.
Epidemiological researches have shown a negative association between cigarette smoking and incidences of PD. In order to investigate the effect of nicotine, one of major cigarette constituents, on blood-brain barrier (BBB) transfer of PD-induced agents, a brain microdialysis technique was used to collect dialysates in the striatum of male Wistar rats treated by MPTP with or without nicotine pretreatment. Saline or single dose of nicotine (0.3, 0.6, 2.0 and 4.0 mg/ kg) was given to rats by intraperitoneal injection 30 min prior to the femoral administration of MPTP (10 mg/ kg, i.v.). Dialysates were collected in a 30-min interval for 150 min. Concentrations of MPTP and MPP+ were quantified by a HPLC method using an ODS-column coupled with a UV-Vis and a fluorescence detector. The results showed that MPTP in 0~30 min interval in brain extracellular fluid can be significantly reduced by nicotine (666.31±65.53 ng/ mL, 561.50±12.11 ng/ mL, 493.23±35.46 ng/ mL, 375.94±76.24 ng/ mL and 329.35±55.14 ng/ mL for control and 0.3, 0.6, 2.0, 4.0 mg/ kg nicotine-pretreatment group, respectively). We also monitored nicotine concentrations in the brain and blood of nicotine at a concentration of 0.6 mg/ kg. The results showed that nicotine concentration in the brain and blood dialysates were 136.8±16.6 ng/ mL and 233.1±9.2 ng/ mL, respectively, in 30~60 min interval. There was no significant difference between a single-dose nicotine treatment and a multi-dose nicotine treatment. In addition, we also investigated whether the inhibitory effects of nicotine on MPTP is gender dependant. As the result, we found that the effect of nicotine showed no significant difference between male and female. Given that tetrahydroisoquinoline (TIQ) is a MPTP-like substance, we also investigated the effect of nicotine on BBB transfer of TIQ. The result indicated TIQ in brain extracellular fluid can not be significantly reduced by nicotine (0.6 and 2.0 mg/ kg).
In conclusion, these data indicate that nicotine can reduce the transfer of MPTP into the brain. However, nicotine can not reduce the transfer of TIQ across BBB. The mechanism of lower incidence of PD associated with smoking needs to be confirmed in the future.

並列關鍵字

microdialysis ； nicotine ； MPTP

參考文獻

[1] K. Abe, T. Saitoh, Y. Horiguchi, I. Utsunomiya, K. Taguchi, Synthesis and neurotoxicity of tetrahydroisoquinoline derivatives for studying Parkinson's disease, Biol. Pharm. Bull. 28 (2005) 1355-1362.

[2] E.D. Abercrombie, J.M. Finaly (Eds.), Monitoring extracellular norepinephrine in brain using in vivo microdialysis and HPLC-CE., In: Robinson, T.E., Justice, Jr J.B. , Microdialysis in the neuroscience, Elsevier, Amsterdam, 1991, 253-274 pp.

[3] L. Antkiewicz-Michaluk, A. Krygowska-Wajs, A. Szczudlik, I. Romanska, J. Vetulani, Increase in salsolinol level in the cerebrospinal fluid of parkinsonian patients is related to dementia: advantage of a new high-performance liquid chromatography methodology, Biol. Psychiatry 42 (1997) 514-518.

[4] S.P. Bagchi, Trace dosages of the neurotoxins MPTP and MPP+ may affect brain dopamine in vivo, Life Sci. 51 (1992) 389-396.

[5] N.L. Benowitz, P. Jacob, 3rd, Daily intake of nicotine during cigarette smoking, Clin. Pharmacol. Ther. 35 (1984) 499-504.