脑摄取平衡时伤害性刺激反应对丙泊酚在犬脊髓不同区域分布的影响
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摘要
背景
全麻药所引起的麻醉状态包含遗忘、意识消失、抑制伤害性刺激反应、制动等多种成分,并且各有其相应的中枢作用区域,脊髓可能是产生抑制伤害性刺激反应的关键部位。丙泊酚具有起效迅速、作用时间短、苏醒快、易控制和副作用少等优点,目前广泛运用于临床麻醉。研究表明其还具有抗外周伤害性刺激反应等作用,主要作用靶位可能在脊髓,但其作用机制尚未明了。丙泊酚中枢麻醉作用有一定的区域选择性,可能是作用于脊髓相应的中枢靶位而发挥其全效应。研究丙泊酚脊髓分布规律有助于了解和揭示其全麻作用机制。
神经和分子生物学的发展为全麻药物作用机制的研究提供了更多的技术和手段,近年来对丙泊酚中枢神经系统作用的研究已经取得一些进展,但目前仍不能阐明其全麻作用机制。中枢神经系统的解剖结构和分布均具有一定的特异性,全麻药发挥其麻醉作用的药理学部位同样具有选择性。在脊髓神经元中存在十几种生物活性物质参与信息的传递,其中促进伤害性信息传递的物质主要有谷氨酸和P物质,抑制伤害性信息传递的物质主要包括阿片肽、GABA和甘氨酸。其中GABA受体是中枢神经系统中最主要的抑制性神经受体,其分布广泛但各有自己的分布区域和不同的药理学及神经电生理学特性。突触学说是全麻作用机制中最重要的学说,该学说认为全麻药物的作用与其影响突触传递的功能有关,主要可能是丙泊酚作用于GABA受体,脊髓胶状质,特别是背角Ⅱ层与Ⅱ、Ⅲ层分界处,GABA能神经元含量丰富,轴突与初级传入终末和深层投射神经元树突形成突触球结构。GABA受体激活后,可增加脊髓神经元电导而产生去极化,从而产生全麻作用。但是,目前对神经系统的了解尚不足,一些研究方法也有其局限性及存在诸多干预因素,已有研究认为外科操作本身可影响脊髓神经元对全麻药的敏感性,因此有必要开拓思路探索丙泊酚在脊髓的摄取和分布规律。
Upton等于1988年首先提出了脑摄取概念及其基本研究方法质量平衡法则(mass balance principles)。药物随血液灌注入脑,由于药物的分布和消除而从血管内分布到血管外进入脑实质的过程称为脑摄取。采用质量平衡法则,通过测量局部器官的动-静脉血药浓度差来计算局部器官摄取药物的量；药物净摄取量为药物经动脉进入器官实质的量；如果药物在器官内不发生代谢,则器官药物浓度为药物净流量与器官质量的比值。基于此法则,上世纪90年代后期有不少学者开始了关于丙泊酚脊髓和脑摄取的研究。Shyr等于1995年报道,以丙泊酚60mg·kg-1·h-1恒速静脉输注时,鼠脊髓和脑组织丙泊酚浓度随静脉输注的时间的增加而增加。最近研究发现,当以丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟时,丙泊酚在犬脑各组织区域的分布趋于一致。丙泊酚脑摄取和分布的实验研究较为深入,而脑摄取平衡时丙泊酚在脊髓组织不同区域的摄取和分布是否一致?伤害性刺激反应对丙泊酚在脊髓不同区域的分布有何影响?以往的实验研究中未曾涉及,有必要进行研究。本研究通过解剖丙泊酚脑摄取达到平衡时的犬脊髓,采用高效液相色谱紫外法(High-pressure liquid chromatography ultra-violet spectroscopy, HPLC-UV)测量脊髓组织不同区域(前角、背角、中间带、前索、后索、外侧索)的丙泊酚浓度,探讨脑摄取平衡时丙泊酚在脊髓不同区域组织的摄取和分布规律,以及伤害性刺激反应对其摄取和分布的影响。
材料和方法
1动物准备与分组：
12只健康犬(实验动物由南方医院动物实验中心提供),雌雄不拘,年龄12-18个月,体重10-12kg,随机分为两组,C组(对照组)和S组(刺激组),每组6只。实验均安排在每天上午9：00至11：00进行。实验前禁食、禁饮12小时。实验时由右后肢大隐静脉建立静脉通路。
2麻醉实施：
C组：静脉注射丙泊酚7mg·kg-1,注射时间为15s,续以70mg·kg-1·h-1恒速静脉输注50分钟。
S组：静脉注射丙泊酚7mg·kg-1,注射时间为15s,续以70mg·kg-1·h-1恒速静脉输注50分钟。当丙泊酚输注45分钟时止血钳上三齿钳夹狗尾正中5min。
两组实验犬达到眼睑反射和脚踏发射消失浅麻醉状态后,经口插入ID9号气管导管,固定并连接呼吸机,吸入纯氧,调节呼吸频率(20-25)次／分,潮气量15ml·kg-1,使呼气末二氧化碳分压(PETCO2)维持在(30-38) mmHg。2%利多卡因浸润麻醉下分离右股动脉,置入20G肝素化套管,接HP多参数监护仪检测平均动脉压(MAP)和脉率(PR)。
3标本采集：
丙泊酚恒速输注50分钟时,2%利多卡因浸润麻醉下快速解剖分离犬右侧颈内动、静脉血管,分别抽取血液2ml快速注入抗凝管中,反复震荡防止血液凝固,立即置于4℃冰箱中保存。取血后立即断头处死实验犬。无菌条件下去除犬椎板。专人解剖分离前角、背角、中间带、前索、后索、外侧索组织适量,去除组织中可见血管,无菌滤纸去除残留血液和脑脊液后分别置于无菌干燥培养皿中,-17℃低温冰箱中保存。
4标本处理：
血标本在4℃低温离心机中离心20min分离血浆和血细胞,转速为10000r·min-1。离心后取血浆200μl置于EP管中,加入乙腈400μl,涡旋器震荡2min。之后再离心10min沉淀血浆蛋白,转速为10000r·min-1。取上清液置于EP管中。标本处理后待测丙泊酚浓度。
脊髓组织标本精确称量(g)后移于匀浆器中,每克脊髓组织中加入乙腈2ml。充分匀浆5min后,匀浆液移于EP管中。匀浆液离心5min,转速10000r·min-1。取上清液置于EP管中。标本处理后待测丙泊酚浓度。
5丙泊酚色谱分析：
采用高效液相色谱紫外(HPLC-UV)-沉淀法测量丙泊酚浓度,外标物为丙泊酚标准品,提取剂为乙腈。流动相：A相为乙酸胺(1mmol·L-1)+乙酸(1g·L-1),B相为甲醇,A：B为25：75,柱温：4℃,自动进样量：20μl,流速：1ml·min-1。检测波长：270nm。
6统计分析：
采用SPSS13.0统计软件包,计量资料以均数±标准差(x±s)表示。组内颈内动脉和静脉血浆丙泊酚浓度比较采用配对t检验,组间丙泊酚血浆浓度比较采用两个独立样本t检验。组内不同脊髓标本中丙泊酚浓度比较采用完全随机设计资料的方差分析(one-way ANOVA),多重比较采用SNK检验；组间相同脊髓部位标本中丙泊酚浓度比较采用两个独立样本t检验。P<0.05为差异有统计学意义。
结果
1麻醉状况：
所有实验动物均安全迅速地达到预定麻醉状态并维持稳定,PR、MAP及PETCO2均波动在正常范围内。C组PR、MAP和PETCO2分别为：80.50±1.38次·min-1、87.83+1.17mmHg和34.83±1.47mmHg。S组刺激前后PR分别为：81.67+1.86次,min-1和94.17±1.94次min-1,刺激后PR明显高于刺激前(t=22.213,P=0.000)；S组刺激前后MAP分别为：88.50±1.05mmHg和101.83±1.72mmHg,刺激后MAP明显高于刺激前(t=13.960,P=0.000)；S组刺激前后PETCO2分别为34.83±1.33mmHg和35.17±1.47mmHg,二者差异无统计学意义(t=0.326,P=0.758)。
2丙泊酚血浆浓度：
C组颈内动脉和颈内静脉血浆丙泊酚浓度分别为5.09±0.03μg/ml和5.07±0.23μg/ml,二者差异无统计学意义(t=0.229,P=0.831)。
S组颈内动脉和颈内静脉血浆丙泊酚浓度分别为5.08±0.03μg/ml和5.03±0.10μg/ml,二者差异无统计学意义(t=1.403,P=0.211)。
C组和S组颈内动脉血浆丙泊酚浓度分别为5.09±0.03μg/ml和5.08±0.03μg/ml,二者差异无统计学意义(t=0.224,P=0.698)。C组和S组颈内静脉血浆丙泊酚浓度分别为5.07+0.23μg／ml和5.03±0.10μg/ml,二者差异无统计学意义(t=1.335,P=0.695)。
3丙泊酚脊髓组织浓度：
C组犬脊髓前角、背角、中间带、前索、后索、外侧索组织丙泊酚浓度(μg·g-1)分别为5.09±0.08、5.10±0.08、5.05±0.19、4.95±0.29、4.99±0.23、5.14±0.45,各区域脊髓组织丙泊酚浓度差异无统计学意义(F=0.469,P=0.798)。
S组犬脊髓前角、背角、中间带、前索、后索、外侧索组织丙泊酚浓度(μg·g-1)分别为：5.14±0.50、7.65±0.47、5.03±0.18、4.94±0.22、7.60±0.82、5.07±0.53,各区域脊髓组织丙泊酚浓度差异有统计学意义(F=41.384,P=0.000),背角和后索丙泊酚浓度明显高于其它脊髓组织(P=0.000)。
脊髓背角丙泊酚浓度S组高于C组(t=13.135,P=0.000),脊髓后索丙泊酚浓度S组高于C组(t=7.447,P=0.000),C、S两组其他相同脊髓区域组织丙泊酚浓度无统计学意义(P>0.05)。
结论
1.HPLC-UV紫外法可用于测定血浆、脑和脊髓组织丙泊酚浓度。
2.丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟,颈动、静脉血药浓度达到平衡状态。
3.丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟,脊髓各区域(前角、背角、中间带、前索、后索、外侧索)丙泊酚分布均衡；给予伤害性刺激后,除脊髓背角和脊髓后索丙泊酚浓度较高外,在其他犬脊髓组织区域(前角、中间带、前索、外侧索)分布均衡。
4.丙泊酚抑制伤害性刺激反应作用的主要作用部位可能在脊髓背角和后索。
Background
The general anesthesia status, induced forgotten, unconsciousness, analgesia, inhibit noxious stimulation-response, braking and other ingredients, has its own the corresponding regions in the central nervous system. The spinal cord might restrain the critical response in the noxious stimulation site. Propofol is a relatively new short-acting intravenous anesthetic with advantage of the quick effect、short duration、prompt revival、easy control and less side effects, which has been widely used in general anesthesia induction and maintenance. Previous studies had shown that propofol played the anti-peripheral role in the reaction of noxious stimulation, the target site of which might located in the spinal cord, but its mechanism had not been understood today. As we known, spinal cord is the effective organ of propofol, and the action of propofol in the spinal cord was discordant in the various region of spinal cord. Therefore, the research of propofol's uptake and distribution in spinal cord is favourable and meaningful to investigate explore the mechanism of general anesthesia.
In the past decade, along with the improvement of theories and techniques of neural and molecular biology, many researches have been done to investigate the anesthetic mechanism of propofol. But its mechanism of general anesthesia was still not able to be explained. The anatomic structure and the distribution in the central nervous system have a certain degree of specificity that the anesthetics have its selective parts to play its narcotic pharmacological effect too. There are dozens of biologically active substances exist in spinal cord neurons mainly involving in the transmission of messages which promote the nociceptive transmission of glutamate and substance P, and inhibit the nociceptive transmission of opioid peptides, GABA and glycine. The GABA receptors in the central nervous system are the major inhibitory receptors, it widespreadly distributed, but had their own regions and the different pharmacological and electrophysiological characteristics to nerves. Synaptic doctrine is the most important doctrine in mechanism of general anesthesia, and it said that the role of anesthesia drugs and their impact was relevant to the function of synaptic transmission, and propofol may be mainly acting on GABA receptors. The GABA neurons are rich in spinal cord substantia gelatinosa, in particular dorsal horn layerⅡⅡ,Ⅲboundary between layers, and axons and primary afferent terminals and synapses ball structure formed by the deep projection neuron dendrites. After GABA receptors were activated, conductance increased as resulting from spinal cord neurons depolarization, and the resulting in general anesthesia effect. But as we all know that our understanding of the nervous system is still inadequate, a number of research methods have its limitations and there are many intervention factors. Many studies suggestted that surgical operations itself can affect the sensitivity of the spinal cord neurons to the anesthetics. Therefore, uptake and regional distribution of propofol in spinal cord must be investigated for understanding its'true anesthetic mechanism.
In 1988, Upton proposed the concept of cerebral uptake and the fundamental research method of mass balancing principles. The process that the drug is filled into the brain with blood and goes into the brain parenchyma from the intravascular which is caused by drug distribution and elimination is known as cerebral uptake.The research about cerebral uptake of propofol was usually baseds on the mass balance principles. By measuring the propofol concentrations in arterial and venous blood of cerebral circulation, we can calculate the cerebral concentrations and evaluate the cerebral uptake of propofol. Based on this rule, many scholars had started the research on the brain uptake of propofol since the late 90s of the last century. In 1995, Shyr reported that propofol concentration in the rat spinal cord and brain tissue increased with the time increased when rats were anesthetized by propofol infusion at a constant rate of 60mg·kg-1·h-1. The recent research had found that when propofol was intravenously infused at a rate of 70mg·kg-1·h-1 to the brain intake balance, the propofol had the same distribution in all regional organizations of the dog brain. The experimental studies about the propofol brain uptaking and the distribution have have been fairly mature. Are the propofol in different regions of spinal cord tissue's uptake and distribution the same? Can the noxious stimulation response impact the distribution of propofol in different regions of the spinal cord? Previous experiments had not been answerd, so it is necessary to do some study.
The aim of this study is to measure propofol concentration of spinal cord by high-pressure liquid chromatography ultra-violet spectroscopy (HPLC-UV) and to investigate the regional distribution of propofol in spinal cord under the circumstandce of noxious stimulation when the cerebral uptake being equilibrium in dogs.
Material and methods
12 healthy dogs aged 12-18 months (male and female) were divided randomly into two groups (group C and group S). All the experiment were scheduled during the day (9:00-11:00) and raised in diet for 12-hour prior to experiment. The venous channel was established in the great saphenous vein of the right posterior limb. Propofol was intravenously injected respectively at a single bolus 7mg·kg-1 in group S and group C in 15 sec. after their eyelid reflex and pedal reflex disappeared, animals were fixed supinely on the platform. The constant intravenous infusion of propofol was taken at a rate of 70 mg·kg-1·h-1 to maintain anesthesia.
When the infusion of propofol was at the 45th min, animals of group S were given stimulation to the end of its tail by hemostat for 5min. Animals of group C were given no stimulation. The blood samples were taken from the right internal carotid and internal jugular vein at the 50th min in group S and at the 50th min in group C. Then the animal was scarificed immediately by decapitation. The frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus of spinal cord were further dissected for determination the concentrations of propofol.
Propofol concentration was determined by HPLC-UV. External standard was a control article of propofol. The analysis was performed with a Shim-pack VP-ODS, 250x4.6mmID, Shim-pack GVP-ODS,10x4.6mmID and a 2996 Waters ultraviolet detector (270nm). The solvent system was purified water-methanol at flow rate of 1ml·min-1.The brain samples were extracted with acetonitrile (2ml·g-1) and homogenized and the blood samples were extracted with acetonitrile (di-volume). After being centrifuged, the supernatant was submitted to HPLC analysis. The sample volume is 20μl.
Measurement data were expressed as mean±standard deviation. All data were analysed with the Statistics Package for Social Sciences (SPSS, version 13.0 for WINDOWS; SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant when P was less than 0.05. We used the Independent-Samples T Test、 Paired-Samples T Test and Repeated Measure to test for differences. Multiple comparisons were analyzed by LSD test. Differences were considered statistically significant when P was less than 0.05.
Results
1. All experimental animals reached safely and quickly the condition of anesthesia and maintained a stable status. In the group C, PETCO2、PR and MAP were 34.83±1.47mmHg、80.50±1.38bpt and 87.83±1.17mmHg respectively. In group S, PETCO2 were 34.83±1.33mmHg and 35.17±1.47mmHg, MAP were 88.50±1.05mmHg and 101.83±1.72mmHg and PR were 81.67±1.86bpt and 94.17±1.94 bpt respectively before and after stimulation, the MAP and PR after stimulation were significantly higher than that before stimulation.
2. The propfol concentration in blood plasma:
The concentration of propofol in internal carotid artery and internal jugular vein blood plasma were 5.09±0.03μg·ml-1,5.07±0.23μg·ml-1 in group C and 5.08±0.03μg·ml-1,5.03±0.10μg·ml-1 in group S respectively and no significant differences.
3. The propofol concentration in spinal cord tissues:
In group S, the differences of propofol concentrations among every region of spinal cord were considered statistically significant (F=41.384, P=0.000),the concentration of propofol in the frontal horn (7.65±0.47)μg·kg-1 and the posterior funiculus (7.60±0.82)μg·kg-1 were higher than that in other region of spinal cord(P<0.05), the differences of propofol concentrations among other region were not considered statistically significant differences(P>0.05). In group C, the differences of propofol concentrations among every region of spinal cord were not considered statistically significant(F= 0.469, P=0.798).
The propofol concentrations of the frontal horn and the posterior funiculus in group S were significantly higher than that in group C (F=1.099,P=0.319).The concentration of propofol in dorsal thalamus and subthalamus were highest under the condition of balance of the brain uptake and noxious stimulation.
Conclusions
1 The HPLC-ultra-violet spectroscopy combining with precipitation method can be used appropriately to determinate the propofol concentration of blood plasma, brain and spinal cord tissues.
2 At 50 min after constant intravenous propofol injection at a rate of 70 mg·kg-1·h-1, plasma propofol concentration reaches equilibrium between internal carotid artery and internal jugular vein, and propofol is distributed evenly among regional spinal cord tissues (frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus) in dogs.
3 Under the circumstance of noxious stimulation, the propofol concentration in frontal horn and posterior funiculus are higher than other region of spinal cord.
全麻药所引起的麻醉状态包含遗忘、意识消失、抑制伤害性刺激反应、制动等多种成分,并且各有其相应的中枢作用区域,脊髓可能是产生抑制伤害性刺激反应的关键部位。丙泊酚具有起效迅速、作用时间短、苏醒快、易控制和副作用少等优点,目前广泛运用于临床麻醉。研究表明其还具有抗外周伤害性刺激反应等作用,主要作用靶位可能在脊髓,但其作用机制尚未明了。丙泊酚中枢麻醉作用有一定的区域选择性,可能是作用于脊髓相应的中枢靶位而发挥其全效应。研究丙泊酚脊髓分布规律有助于了解和揭示其全麻作用机制。
神经和分子生物学的发展为全麻药物作用机制的研究提供了更多的技术和手段,近年来对丙泊酚中枢神经系统作用的研究已经取得一些进展,但目前仍不能阐明其全麻作用机制。中枢神经系统的解剖结构和分布均具有一定的特异性,全麻药发挥其麻醉作用的药理学部位同样具有选择性。在脊髓神经元中存在十几种生物活性物质参与信息的传递,其中促进伤害性信息传递的物质主要有谷氨酸和P物质,抑制伤害性信息传递的物质主要包括阿片肽、GABA和甘氨酸。其中GABA受体是中枢神经系统中最主要的抑制性神经受体,其分布广泛但各有自己的分布区域和不同的药理学及神经电生理学特性。突触学说是全麻作用机制中最重要的学说,该学说认为全麻药物的作用与其影响突触传递的功能有关,主要可能是丙泊酚作用于GABA受体,脊髓胶状质,特别是背角Ⅱ层与Ⅱ、Ⅲ层分界处,GABA能神经元含量丰富,轴突与初级传入终末和深层投射神经元树突形成突触球结构。GABA受体激活后,可增加脊髓神经元电导而产生去极化,从而产生全麻作用。但是,目前对神经系统的了解尚不足,一些研究方法也有其局限性及存在诸多干预因素,已有研究认为外科操作本身可影响脊髓神经元对全麻药的敏感性,因此有必要开拓思路探索丙泊酚在脊髓的摄取和分布规律。
Upton等于1988年首先提出了脑摄取概念及其基本研究方法质量平衡法则(mass balance principles)。药物随血液灌注入脑,由于药物的分布和消除而从血管内分布到血管外进入脑实质的过程称为脑摄取。采用质量平衡法则,通过测量局部器官的动-静脉血药浓度差来计算局部器官摄取药物的量；药物净摄取量为药物经动脉进入器官实质的量；如果药物在器官内不发生代谢,则器官药物浓度为药物净流量与器官质量的比值。基于此法则,上世纪90年代后期有不少学者开始了关于丙泊酚脊髓和脑摄取的研究。Shyr等于1995年报道,以丙泊酚60mg·kg-1·h-1恒速静脉输注时,鼠脊髓和脑组织丙泊酚浓度随静脉输注的时间的增加而增加。最近研究发现,当以丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟时,丙泊酚在犬脑各组织区域的分布趋于一致。丙泊酚脑摄取和分布的实验研究较为深入,而脑摄取平衡时丙泊酚在脊髓组织不同区域的摄取和分布是否一致?伤害性刺激反应对丙泊酚在脊髓不同区域的分布有何影响?以往的实验研究中未曾涉及,有必要进行研究。本研究通过解剖丙泊酚脑摄取达到平衡时的犬脊髓,采用高效液相色谱紫外法(High-pressure liquid chromatography ultra-violet spectroscopy, HPLC-UV)测量脊髓组织不同区域(前角、背角、中间带、前索、后索、外侧索)的丙泊酚浓度,探讨脑摄取平衡时丙泊酚在脊髓不同区域组织的摄取和分布规律,以及伤害性刺激反应对其摄取和分布的影响。
材料和方法
1动物准备与分组：
12只健康犬(实验动物由南方医院动物实验中心提供),雌雄不拘,年龄12-18个月,体重10-12kg,随机分为两组,C组(对照组)和S组(刺激组),每组6只。实验均安排在每天上午9：00至11：00进行。实验前禁食、禁饮12小时。实验时由右后肢大隐静脉建立静脉通路。
2麻醉实施：
C组：静脉注射丙泊酚7mg·kg-1,注射时间为15s,续以70mg·kg-1·h-1恒速静脉输注50分钟。
S组：静脉注射丙泊酚7mg·kg-1,注射时间为15s,续以70mg·kg-1·h-1恒速静脉输注50分钟。当丙泊酚输注45分钟时止血钳上三齿钳夹狗尾正中5min。
两组实验犬达到眼睑反射和脚踏发射消失浅麻醉状态后,经口插入ID9号气管导管,固定并连接呼吸机,吸入纯氧,调节呼吸频率(20-25)次／分,潮气量15ml·kg-1,使呼气末二氧化碳分压(PETCO2)维持在(30-38) mmHg。2%利多卡因浸润麻醉下分离右股动脉,置入20G肝素化套管,接HP多参数监护仪检测平均动脉压(MAP)和脉率(PR)。
3标本采集：
丙泊酚恒速输注50分钟时,2%利多卡因浸润麻醉下快速解剖分离犬右侧颈内动、静脉血管,分别抽取血液2ml快速注入抗凝管中,反复震荡防止血液凝固,立即置于4℃冰箱中保存。取血后立即断头处死实验犬。无菌条件下去除犬椎板。专人解剖分离前角、背角、中间带、前索、后索、外侧索组织适量,去除组织中可见血管,无菌滤纸去除残留血液和脑脊液后分别置于无菌干燥培养皿中,-17℃低温冰箱中保存。
4标本处理：
血标本在4℃低温离心机中离心20min分离血浆和血细胞,转速为10000r·min-1。离心后取血浆200μl置于EP管中,加入乙腈400μl,涡旋器震荡2min。之后再离心10min沉淀血浆蛋白,转速为10000r·min-1。取上清液置于EP管中。标本处理后待测丙泊酚浓度。
脊髓组织标本精确称量(g)后移于匀浆器中,每克脊髓组织中加入乙腈2ml。充分匀浆5min后,匀浆液移于EP管中。匀浆液离心5min,转速10000r·min-1。取上清液置于EP管中。标本处理后待测丙泊酚浓度。
5丙泊酚色谱分析：
采用高效液相色谱紫外(HPLC-UV)-沉淀法测量丙泊酚浓度,外标物为丙泊酚标准品,提取剂为乙腈。流动相：A相为乙酸胺(1mmol·L-1)+乙酸(1g·L-1),B相为甲醇,A：B为25：75,柱温：4℃,自动进样量：20μl,流速：1ml·min-1。检测波长：270nm。
6统计分析：
采用SPSS13.0统计软件包,计量资料以均数±标准差(x±s)表示。组内颈内动脉和静脉血浆丙泊酚浓度比较采用配对t检验,组间丙泊酚血浆浓度比较采用两个独立样本t检验。组内不同脊髓标本中丙泊酚浓度比较采用完全随机设计资料的方差分析(one-way ANOVA),多重比较采用SNK检验；组间相同脊髓部位标本中丙泊酚浓度比较采用两个独立样本t检验。P<0.05为差异有统计学意义。
结果
1麻醉状况：
所有实验动物均安全迅速地达到预定麻醉状态并维持稳定,PR、MAP及PETCO2均波动在正常范围内。C组PR、MAP和PETCO2分别为：80.50±1.38次·min-1、87.83+1.17mmHg和34.83±1.47mmHg。S组刺激前后PR分别为：81.67+1.86次,min-1和94.17±1.94次min-1,刺激后PR明显高于刺激前(t=22.213,P=0.000)；S组刺激前后MAP分别为：88.50±1.05mmHg和101.83±1.72mmHg,刺激后MAP明显高于刺激前(t=13.960,P=0.000)；S组刺激前后PETCO2分别为34.83±1.33mmHg和35.17±1.47mmHg,二者差异无统计学意义(t=0.326,P=0.758)。
2丙泊酚血浆浓度：
C组颈内动脉和颈内静脉血浆丙泊酚浓度分别为5.09±0.03μg/ml和5.07±0.23μg/ml,二者差异无统计学意义(t=0.229,P=0.831)。
S组颈内动脉和颈内静脉血浆丙泊酚浓度分别为5.08±0.03μg/ml和5.03±0.10μg/ml,二者差异无统计学意义(t=1.403,P=0.211)。
C组和S组颈内动脉血浆丙泊酚浓度分别为5.09±0.03μg/ml和5.08±0.03μg/ml,二者差异无统计学意义(t=0.224,P=0.698)。C组和S组颈内静脉血浆丙泊酚浓度分别为5.07+0.23μg／ml和5.03±0.10μg/ml,二者差异无统计学意义(t=1.335,P=0.695)。
3丙泊酚脊髓组织浓度：
C组犬脊髓前角、背角、中间带、前索、后索、外侧索组织丙泊酚浓度(μg·g-1)分别为5.09±0.08、5.10±0.08、5.05±0.19、4.95±0.29、4.99±0.23、5.14±0.45,各区域脊髓组织丙泊酚浓度差异无统计学意义(F=0.469,P=0.798)。
S组犬脊髓前角、背角、中间带、前索、后索、外侧索组织丙泊酚浓度(μg·g-1)分别为：5.14±0.50、7.65±0.47、5.03±0.18、4.94±0.22、7.60±0.82、5.07±0.53,各区域脊髓组织丙泊酚浓度差异有统计学意义(F=41.384,P=0.000),背角和后索丙泊酚浓度明显高于其它脊髓组织(P=0.000)。
脊髓背角丙泊酚浓度S组高于C组(t=13.135,P=0.000),脊髓后索丙泊酚浓度S组高于C组(t=7.447,P=0.000),C、S两组其他相同脊髓区域组织丙泊酚浓度无统计学意义(P>0.05)。
结论
1.HPLC-UV紫外法可用于测定血浆、脑和脊髓组织丙泊酚浓度。
2.丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟,颈动、静脉血药浓度达到平衡状态。
3.丙泊酚70mg·kg-1·h-1恒速静脉输注50分钟,脊髓各区域(前角、背角、中间带、前索、后索、外侧索)丙泊酚分布均衡；给予伤害性刺激后,除脊髓背角和脊髓后索丙泊酚浓度较高外,在其他犬脊髓组织区域(前角、中间带、前索、外侧索)分布均衡。
4.丙泊酚抑制伤害性刺激反应作用的主要作用部位可能在脊髓背角和后索。
Background
The general anesthesia status, induced forgotten, unconsciousness, analgesia, inhibit noxious stimulation-response, braking and other ingredients, has its own the corresponding regions in the central nervous system. The spinal cord might restrain the critical response in the noxious stimulation site. Propofol is a relatively new short-acting intravenous anesthetic with advantage of the quick effect、short duration、prompt revival、easy control and less side effects, which has been widely used in general anesthesia induction and maintenance. Previous studies had shown that propofol played the anti-peripheral role in the reaction of noxious stimulation, the target site of which might located in the spinal cord, but its mechanism had not been understood today. As we known, spinal cord is the effective organ of propofol, and the action of propofol in the spinal cord was discordant in the various region of spinal cord. Therefore, the research of propofol's uptake and distribution in spinal cord is favourable and meaningful to investigate explore the mechanism of general anesthesia.
In the past decade, along with the improvement of theories and techniques of neural and molecular biology, many researches have been done to investigate the anesthetic mechanism of propofol. But its mechanism of general anesthesia was still not able to be explained. The anatomic structure and the distribution in the central nervous system have a certain degree of specificity that the anesthetics have its selective parts to play its narcotic pharmacological effect too. There are dozens of biologically active substances exist in spinal cord neurons mainly involving in the transmission of messages which promote the nociceptive transmission of glutamate and substance P, and inhibit the nociceptive transmission of opioid peptides, GABA and glycine. The GABA receptors in the central nervous system are the major inhibitory receptors, it widespreadly distributed, but had their own regions and the different pharmacological and electrophysiological characteristics to nerves. Synaptic doctrine is the most important doctrine in mechanism of general anesthesia, and it said that the role of anesthesia drugs and their impact was relevant to the function of synaptic transmission, and propofol may be mainly acting on GABA receptors. The GABA neurons are rich in spinal cord substantia gelatinosa, in particular dorsal horn layerⅡⅡ,Ⅲboundary between layers, and axons and primary afferent terminals and synapses ball structure formed by the deep projection neuron dendrites. After GABA receptors were activated, conductance increased as resulting from spinal cord neurons depolarization, and the resulting in general anesthesia effect. But as we all know that our understanding of the nervous system is still inadequate, a number of research methods have its limitations and there are many intervention factors. Many studies suggestted that surgical operations itself can affect the sensitivity of the spinal cord neurons to the anesthetics. Therefore, uptake and regional distribution of propofol in spinal cord must be investigated for understanding its'true anesthetic mechanism.
In 1988, Upton proposed the concept of cerebral uptake and the fundamental research method of mass balancing principles. The process that the drug is filled into the brain with blood and goes into the brain parenchyma from the intravascular which is caused by drug distribution and elimination is known as cerebral uptake.The research about cerebral uptake of propofol was usually baseds on the mass balance principles. By measuring the propofol concentrations in arterial and venous blood of cerebral circulation, we can calculate the cerebral concentrations and evaluate the cerebral uptake of propofol. Based on this rule, many scholars had started the research on the brain uptake of propofol since the late 90s of the last century. In 1995, Shyr reported that propofol concentration in the rat spinal cord and brain tissue increased with the time increased when rats were anesthetized by propofol infusion at a constant rate of 60mg·kg-1·h-1. The recent research had found that when propofol was intravenously infused at a rate of 70mg·kg-1·h-1 to the brain intake balance, the propofol had the same distribution in all regional organizations of the dog brain. The experimental studies about the propofol brain uptaking and the distribution have have been fairly mature. Are the propofol in different regions of spinal cord tissue's uptake and distribution the same? Can the noxious stimulation response impact the distribution of propofol in different regions of the spinal cord? Previous experiments had not been answerd, so it is necessary to do some study.
The aim of this study is to measure propofol concentration of spinal cord by high-pressure liquid chromatography ultra-violet spectroscopy (HPLC-UV) and to investigate the regional distribution of propofol in spinal cord under the circumstandce of noxious stimulation when the cerebral uptake being equilibrium in dogs.
Material and methods
12 healthy dogs aged 12-18 months (male and female) were divided randomly into two groups (group C and group S). All the experiment were scheduled during the day (9:00-11:00) and raised in diet for 12-hour prior to experiment. The venous channel was established in the great saphenous vein of the right posterior limb. Propofol was intravenously injected respectively at a single bolus 7mg·kg-1 in group S and group C in 15 sec. after their eyelid reflex and pedal reflex disappeared, animals were fixed supinely on the platform. The constant intravenous infusion of propofol was taken at a rate of 70 mg·kg-1·h-1 to maintain anesthesia.
When the infusion of propofol was at the 45th min, animals of group S were given stimulation to the end of its tail by hemostat for 5min. Animals of group C were given no stimulation. The blood samples were taken from the right internal carotid and internal jugular vein at the 50th min in group S and at the 50th min in group C. Then the animal was scarificed immediately by decapitation. The frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus of spinal cord were further dissected for determination the concentrations of propofol.
Propofol concentration was determined by HPLC-UV. External standard was a control article of propofol. The analysis was performed with a Shim-pack VP-ODS, 250x4.6mmID, Shim-pack GVP-ODS,10x4.6mmID and a 2996 Waters ultraviolet detector (270nm). The solvent system was purified water-methanol at flow rate of 1ml·min-1.The brain samples were extracted with acetonitrile (2ml·g-1) and homogenized and the blood samples were extracted with acetonitrile (di-volume). After being centrifuged, the supernatant was submitted to HPLC analysis. The sample volume is 20μl.
Measurement data were expressed as mean±standard deviation. All data were analysed with the Statistics Package for Social Sciences (SPSS, version 13.0 for WINDOWS; SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant when P was less than 0.05. We used the Independent-Samples T Test、 Paired-Samples T Test and Repeated Measure to test for differences. Multiple comparisons were analyzed by LSD test. Differences were considered statistically significant when P was less than 0.05.
Results
1. All experimental animals reached safely and quickly the condition of anesthesia and maintained a stable status. In the group C, PETCO2、PR and MAP were 34.83±1.47mmHg、80.50±1.38bpt and 87.83±1.17mmHg respectively. In group S, PETCO2 were 34.83±1.33mmHg and 35.17±1.47mmHg, MAP were 88.50±1.05mmHg and 101.83±1.72mmHg and PR were 81.67±1.86bpt and 94.17±1.94 bpt respectively before and after stimulation, the MAP and PR after stimulation were significantly higher than that before stimulation.
2. The propfol concentration in blood plasma:
The concentration of propofol in internal carotid artery and internal jugular vein blood plasma were 5.09±0.03μg·ml-1,5.07±0.23μg·ml-1 in group C and 5.08±0.03μg·ml-1,5.03±0.10μg·ml-1 in group S respectively and no significant differences.
3. The propofol concentration in spinal cord tissues:
In group S, the differences of propofol concentrations among every region of spinal cord were considered statistically significant (F=41.384, P=0.000),the concentration of propofol in the frontal horn (7.65±0.47)μg·kg-1 and the posterior funiculus (7.60±0.82)μg·kg-1 were higher than that in other region of spinal cord(P<0.05), the differences of propofol concentrations among other region were not considered statistically significant differences(P>0.05). In group C, the differences of propofol concentrations among every region of spinal cord were not considered statistically significant(F= 0.469, P=0.798).
The propofol concentrations of the frontal horn and the posterior funiculus in group S were significantly higher than that in group C (F=1.099,P=0.319).The concentration of propofol in dorsal thalamus and subthalamus were highest under the condition of balance of the brain uptake and noxious stimulation.
Conclusions
1 The HPLC-ultra-violet spectroscopy combining with precipitation method can be used appropriately to determinate the propofol concentration of blood plasma, brain and spinal cord tissues.
2 At 50 min after constant intravenous propofol injection at a rate of 70 mg·kg-1·h-1, plasma propofol concentration reaches equilibrium between internal carotid artery and internal jugular vein, and propofol is distributed evenly among regional spinal cord tissues (frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus) in dogs.
3 Under the circumstance of noxious stimulation, the propofol concentration in frontal horn and posterior funiculus are higher than other region of spinal cord.
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[15]Shyr MH, Tsai TH, Tan PP, et al, Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat[J].Neurosci Lett,1995,30,184(3):212-5.
[16]Dowrie RH, Ebling WF, Mandema JW, et al. High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissue using electrochemical detection[J]. J Chromatogr B Biomed Appl,1996, 678(2):279-88.
[17]林春水,卢刚,古妙宁,等.静脉注射异丙酚在犬脑的摄取和分布[J].中华麻醉学杂志,2007,27：969-971.
[18]林春水,刘长涛,古妙宁,等.不同麻醉深度下异丙酚在犬脑组织的摄取和分布[J].广东医学,2008,29：750-752.
[19]林春水,卢刚,古妙宁,等.脑摄取平衡时异丙酚在犬脑不同区域的分布[J].南方医科大学学报,2007,27(6)：836-838.
[20]古妙宁,盖成林,林春水,等.恒速静脉输注异丙酚时脑摄取的研究[J]. 中华麻醉学杂志,2001,21(3)：133-5.
[21]Riu PL, Riu G, Testa C,et al. Disposition of propofol between red blood cell, plasma brain and cerebrospinal fluid in rabbits[J]. Eur J Anaesthesiol 2000; 17(1):18-22.
[22]Larsson JE,Wahlstron GThe influence of age and administration rate on the brain sensitivity to propofol in rats[J] Acta Anaesthesiol Scand,1998,42(8): 987-994.
[23]许雄伟,王长进.异丙酚生物样品的测定方法及药代动力学研究[J].海峡药学,2002,14(60)：5-7.
[24]Dawidowicz AL, Fijalkowska A, Determination of propofol in blood by HPLC. Comparison of the extraction and precipitation methods[J]. Chromatogr Sci 1995;33(7);377-82.
[25]王增寿,金胜威,朱光辉,等.高效液相色谱荧光法测定脑脊液中丙泊酚浓度[J].中国现代应用药学杂志,2004,21(5)：405-6.
[26]Upton RN, Ludbrook GL. A model of the kinetics and dynamics of induction of anaesthesia in sheep:variable estimation for thiopental and comparison with propofol[J]. Br J Anaesth.1999;82(6):890.
[27]Giuseppe Trapani, Cosimo Altomare, Enrico Sanna, et al. Propofol in anesthesia. Mechanism of action, structure-activity relationships, and drug delivery[J]. Current medicinal chemistry,2000,7:249-271.
[28]Short CE, Bufalari A. Propofol anesthesia. Vet Clin North Am Small Anim Pract [J].1999 May; 29 (3):747-748.
[29]欧阳铭文,古妙宁,林春水,等.效应室浓度作为目标浓度靶控输注异丙酚的脑摄取研究[J].第一军医大学学报,2002,22(1)：642-646.
[30]Rampil, IJ, Mason. Anesthetic potency (MAC) is independent of forebrain structures in the rat[J]. Anesthesiology,1993,78:707-712.
[31]Joo DT, Xiong Z, MacDonald JF, et al. Blockade of glutamate receptors and barbiturate anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice[J]. Anesthesiology,1999,91:1329-1341.
[32]Bryson HM,Fulton BR,Fawds D,et al. Propofol:an update of its use in anesthesia and conscious sedation[J].Drugs,1995,50:513-559.
[33]Antognini JF. The relationship among brain, spinal cord and anesthetic requirements[J].Med hypothese,1997,48:83-87.
[34]Jewett BA, Gibbs LM, Tarasiuk A, et al. Propofol and barbiturate depression of spinal nociceptive neurotransmission[J]. Anesthesiology,1992,77:1148-1154.
[35]Rampil IJ, King BS, Volatile anesthetics depress motor neurons [J]. Anesthesiology,1996,85:129.
[36]Pereon Y, Bernard JM, Nguyen, Tich S, et al. The effect of desfluran on the nervous system:from spinal cord to muscles [J]. Anesth-Analg,1999,89 (2):490.
[37]Guertin PA, Hounsgaard J. Nonvolatile general anaesthetics reduce spinal activity by suppressing plateau potentials[J].Neuroscience,1999,88:353-358.
[38]Zhou HH, Jin TT, Qin B, et al. Suppression of spinal cord motoneuron excitability correlates with surgical immobility during isoflurane anesthesia [J].Anesthesiology,1998,88:955-961.
[39]林春水,谢丰永,古妙宁,等.颈内动、静脉血药浓度平衡时异丙酚在犬脑不同区域的摄取和分布[J].南方医科大学学报,2009,29(2)：242-245.
[40]Trapani G, Altomare C, Liso G, et al. Propofol in anesthesia. Mechanism of action, structureactivity relationships, and drug delivery[J]. Currmed chem, 2000,7(2):249-271.
[41]Watanabe H, Nakayama D, YuhkiM. Differential inhibitory effects of muopioids on substance P2 and capsaicin induced nociceptive behavior in mice [J]. Pep tides,2006,27(4):760-768.
[42]Diaz Ruiz A, Salgado Ceballos H, Montes S. Acute alterations of glutamine, GABA, and other aminoacids after spinal cord contusion in rats[J]. Neurochem Res,2007,32(1):57-63.
[43]Maeda Y, Lisi TL, Vance CG Release of GABA and activation of GABA (A) in t he spinal cord mediates the effects of TENS in rats [J].Brain Res, 2007,1136(1):43-50.
[44]Rodicio MC, Verona VC, Anton BI, et al. Coloalization of dopamine and GABA in spinal cord neurons in the sea lamprey [J]. Brain research bulletin, 2008,76:45-49.
[45]King BS, Rampil IJ. Anesthetic depression of spinal motor neurons may contribute to lack of movement in response to noxious stimuli [J]. Anesthesiology,1994,81:1484.
[46]Zhou HH, Turndorf H. Hyper- and hypoventilation affects spinal motor neuron excitability during isoflurane anesthesia [J]. Anestha-Analg,1998,87:407.
[1]林春水,于冬男,古妙宁,等.异丙酚靶控输注对健康志愿者脑葡萄糖代谢的影响[J].中华麻醉学杂志.2004,24(9)：645-648.
[2]Minoru M, Kurumi S, Shiro H, et al. GABA receptor activation in the lumbosacral spinal cord decreases detrusor overactivity in spinal cord injured rats[J].The journal of urology,2008,179(5):1178-1183.
[3]Xu AJ, Duan SM, Zeng YM. Effect of intrathecal NMDA and AMPA receptors agonists or antagonists on antinociception of propofol[J]. Acta Pharmacol Sin, 2004,25:9-14.
[4]Rampil, IJ, Mason. Anesthetic potency (MAC) is independent of forebrain structures in the rat[J]. Anesthesiology,1993,78:707-712.
[5]Nicoll RA, Madison DV. General anesthetics hyperpolarize neurons in the vertebrate central nervous system[J]. Science Magazine,1982,217:1055-1057.
[6]Hesdley PM, Parsons, West DC. Opioid receptor-mediated effects on spinal responses to controlled noxious[J]. Fine afferent nerve fibres and pain, 1987,25:620-624.
[7]Rampil IJ, King BS, Volatile anesthetics depress motor neurons[J]. Anesthesiology, 1996,85:129.
[8]Zhou HH, Turndorf H. Hyper- and hypoventilation affects spinal motor neuron excitability during isoflurane anesthesia[J]. Anestha-Analg,1998,87:407.
[9]Sudo M, Sudo S, Chen XG, et al. Thiopental directly depresses lumbar dorsal horn neuronal responses to noxious mechanical stimulation in goats [J]. Acta Anaesthesiol Scand,2001,45:823-829.
[10]Yamamori Y, Kishikawa K,Collins JG. Halothane effects on low-threshold receptive field size of rat spinal dorsal horn neuron appear to be independent of supraspinal modulatory systems [J]. Brain Res,1995;702:162-168.
[11]每晓鹏,王智明,张惠,等。氯胺酮对大鼠脊髓背角胶状质内对突触前神经递质释放的影响[J].神经解剖学杂志.2007,23(3)：239-244.
[12]Jurna L. Depression of nociceptive sensory activity in the rat spinal cord due to the intrathecal administration of drugs:effect of diazepam [J]. Neurosurgery, 1984,15(6):917-20.
[13]Hanzawa, Shinji. Midazolam presynaptically inhibits excitatory synaptic transmission in the superficial dorsal horn of the rat spinal cord [J]. Japanese Journal of Anesthesiology,1999,48(5):474-480.
[14]Cheng G, Kendig JJ. Enflurane decreases glutamate neurotransmission to spinal cord motor neurons by both presynaptic and postsynaptic actions[J]. Anesth Analg,2003,96:1354～1359.
[15]Wong SM, Fong E, Tauck DL, et al. Ethanol as a general anesthetic:actions in spinal cord [J]. Eur J Pharmacol,1997,329:121～127.
[16]Wong SM, Cheng G, Homanics GE, et al. Enflurane actionson spinal cords from mice that lack the beta3 subunit of the GABA(A) receptor [J]. Anesthesiology,2001,95:154～164.
[17]Bohlhalter SM, Hanns M, Jean MF, Inhibitory neurotransmission in rat spinal cord:co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining [J].Brain reseatch,1994,642(11):59-69.
[18]Dong XP, Xu TL. The actions of propofol on gammaaminobutyric acid2A and glycine receptors in acutely dissociated spinal dorsal horn neurons of the rat[J]. Anesth Analg,2002,95:907～914.
[19]Nadeson R, Goodchild CS.Antinociceptive Properties of Propofol: Involvement of Spinal Cord y-Aminobutyric Acid A Receptors [J]. Journal of Pharmacology and Experimental,1997,21(5):210-221.
[20]Sonner JM, Antognini JF, Dutton RC, et al. Inhaled anesthetics and immobility:mechanisms, mysteries, and minimum alveolar anesthetic concentration[J]. Anesth Analg,2003,97:719～740.
[21]Savola MK, Woodley SJ, Kendig JJ. Isoflurane depresses both glutamate- and peptide-mediated slow synaptic transmission in neonatal rat spinal cord[J]. ACAD Sci,1991,625:281-2.
[22]Takenoshita M,Steinbach JH.Halothane blocks low-voltage-activated calcium current in rate sensory neurons[J].Journal of Neuroscience,1991,11:1404-1412.
[23]Wang MY, Rampil IJ, Kendig JJ. Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors[J]. J Pharmacol Exp Ther,1999, 290:362-367.
[24]Ren SL, Wang MY. Supressive effects of propofol on neonate rat spinal cord motoneurons in vitro[J]. Soc Neurosci Abstr,2002,28:446.6.
[25]Asai T, Kusudo K, Ikeda H, et al. Effects of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices:evidence for a presynaptic action[J]. Eur J Neurosci,2002,15:1278～1290.
[26]麻海春,冯春生,曾维安,等.电压依赖性K+通道在大鼠腺苷脑和脊髓镇痛及氟烷麻醉中的中用[J].中华麻醉学杂志,2007,27(4)：373-375.
[2]Anker-Moller E, Spangsberg N, Arendt-Nielsen L, et al. Subhypnotic dose of thiopentone and propofol cause analgesia in experimentally induced acute pain. Br J Anaesth,1991,66(2):185-188.
[3]Minoru M, Kurumi S, Shiro H, et al. GABA receptor activation in the lumbosacral spinal cord decreases detrusor overactivity in spinal cord injured rats [J]. The journal of urology,2008,179(5):1178-1183.
[4]Antogini JF, Carstens E. In vivo characterization of clinical anesthesia and its components [J]. Br J Anaesth,2002,89,156-166.
[5]Nadeson R, Goodchild CS. Antinociceptive properties of propofol: involvement of spinal cord gamma-aminobutyric acid(A) receptors[J].J Pharmacol Exp Ther,1997,282(3):1181-1186.
[6]Xu AJ,Duan SM,Zeng YM. Effect of intrathecal NMD A and AMPA receptors agonists or antagonists on antinociception of propofol [J]. Acta Pharmacol Sin, 2004,25(1):9-14.
[7]Ratnakumari L, Hemmings HC. Effects of propofol on sodium channeldependent sodium influx and glutanmate release in rat cerebrocortical synaptosomes[J]. Anesthesiology,1997,86(2):428-439.
[8]Wang QY, Cao JL, Zeng YM, et al. GABAA receptors partially mediated propo-induced hyperalgesia at superspinal level and analgesia at spinal cord level in rats[J]. Acta Pharmacol Sin.2004,25(12):1619-1625.
[9]Petersen-Felix S, Arendt-Nielsen L, Bak P, et al. Psychophysical and electrophysiological responses to experimental pain may be influenced by sedation:comparison of the effects of a hypnotic (propofol) and an analgesic (alfentanil) [J]. Br J Anesth,1996,77(2):165-171.
[10]Eccles JC, Schade JP. Organization of the spinal cord [M], Amsterdam; New York:Elsevier Bub. Co,1964:58-62.
[11]Renshaw B. Central effects of centripetal impulse in axons of spinal ventral roots [J]. J Neutophysiol.1964,9:191-204.
[12]Guertin PA, Hounsgaard J. Non-volatile general anaesthetice reduce spinal activtity by suppressing plateau potentials[J]. Neuroscience,1994,88:353.
[13]Upton RN, Mather LE, Runciman WB, et al. The use of mass balance principles to describe regional drug distribution and elimination[J]. J Pharmacokinet Biopharm.1988,16(1):13-29.
[14]Huq CC Jr. Pharmacokinetics of drugs administered intravenously [J].Anesth Analg,1978,57:704-723.
[15]Shyr MH, Tsai TH, Tan PP, et al, Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat[J].Neurosci Lett,1995,30,184(3):212-5.
[16]Dowrie RH, Ebling WF, Mandema JW, et al. High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissue using electrochemical detection[J]. J Chromatogr B Biomed Appl,1996, 678(2):279-88.
[17]林春水,卢刚,古妙宁,等.静脉注射异丙酚在犬脑的摄取和分布[J].中华麻醉学杂志,2007,27：969-971.
[18]林春水,刘长涛,古妙宁,等.不同麻醉深度下异丙酚在犬脑组织的摄取和分布[J].广东医学,2008,29：750-752.
[19]林春水,卢刚,古妙宁,等.脑摄取平衡时异丙酚在犬脑不同区域的分布[J].南方医科大学学报,2007,27(6)：836-838.
[20]古妙宁,盖成林,林春水,等.恒速静脉输注异丙酚时脑摄取的研究[J]. 中华麻醉学杂志,2001,21(3)：133-5.
[21]Riu PL, Riu G, Testa C,et al. Disposition of propofol between red blood cell, plasma brain and cerebrospinal fluid in rabbits[J]. Eur J Anaesthesiol 2000; 17(1):18-22.
[22]Larsson JE,Wahlstron GThe influence of age and administration rate on the brain sensitivity to propofol in rats[J] Acta Anaesthesiol Scand,1998,42(8): 987-994.
[23]许雄伟,王长进.异丙酚生物样品的测定方法及药代动力学研究[J].海峡药学,2002,14(60)：5-7.
[24]Dawidowicz AL, Fijalkowska A, Determination of propofol in blood by HPLC. Comparison of the extraction and precipitation methods[J]. Chromatogr Sci 1995;33(7);377-82.
[25]王增寿,金胜威,朱光辉,等.高效液相色谱荧光法测定脑脊液中丙泊酚浓度[J].中国现代应用药学杂志,2004,21(5)：405-6.
[26]Upton RN, Ludbrook GL. A model of the kinetics and dynamics of induction of anaesthesia in sheep:variable estimation for thiopental and comparison with propofol[J]. Br J Anaesth.1999;82(6):890.
[27]Giuseppe Trapani, Cosimo Altomare, Enrico Sanna, et al. Propofol in anesthesia. Mechanism of action, structure-activity relationships, and drug delivery[J]. Current medicinal chemistry,2000,7:249-271.
[28]Short CE, Bufalari A. Propofol anesthesia. Vet Clin North Am Small Anim Pract [J].1999 May; 29 (3):747-748.
[29]欧阳铭文,古妙宁,林春水,等.效应室浓度作为目标浓度靶控输注异丙酚的脑摄取研究[J].第一军医大学学报,2002,22(1)：642-646.
[30]Rampil, IJ, Mason. Anesthetic potency (MAC) is independent of forebrain structures in the rat[J]. Anesthesiology,1993,78:707-712.
[31]Joo DT, Xiong Z, MacDonald JF, et al. Blockade of glutamate receptors and barbiturate anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice[J]. Anesthesiology,1999,91:1329-1341.
[32]Bryson HM,Fulton BR,Fawds D,et al. Propofol:an update of its use in anesthesia and conscious sedation[J].Drugs,1995,50:513-559.
[33]Antognini JF. The relationship among brain, spinal cord and anesthetic requirements[J].Med hypothese,1997,48:83-87.
[34]Jewett BA, Gibbs LM, Tarasiuk A, et al. Propofol and barbiturate depression of spinal nociceptive neurotransmission[J]. Anesthesiology,1992,77:1148-1154.
[35]Rampil IJ, King BS, Volatile anesthetics depress motor neurons [J]. Anesthesiology,1996,85:129.
[36]Pereon Y, Bernard JM, Nguyen, Tich S, et al. The effect of desfluran on the nervous system:from spinal cord to muscles [J]. Anesth-Analg,1999,89 (2):490.
[37]Guertin PA, Hounsgaard J. Nonvolatile general anaesthetics reduce spinal activity by suppressing plateau potentials[J].Neuroscience,1999,88:353-358.
[38]Zhou HH, Jin TT, Qin B, et al. Suppression of spinal cord motoneuron excitability correlates with surgical immobility during isoflurane anesthesia [J].Anesthesiology,1998,88:955-961.
[39]林春水,谢丰永,古妙宁,等.颈内动、静脉血药浓度平衡时异丙酚在犬脑不同区域的摄取和分布[J].南方医科大学学报,2009,29(2)：242-245.
[40]Trapani G, Altomare C, Liso G, et al. Propofol in anesthesia. Mechanism of action, structureactivity relationships, and drug delivery[J]. Currmed chem, 2000,7(2):249-271.
[41]Watanabe H, Nakayama D, YuhkiM. Differential inhibitory effects of muopioids on substance P2 and capsaicin induced nociceptive behavior in mice [J]. Pep tides,2006,27(4):760-768.
[42]Diaz Ruiz A, Salgado Ceballos H, Montes S. Acute alterations of glutamine, GABA, and other aminoacids after spinal cord contusion in rats[J]. Neurochem Res,2007,32(1):57-63.
[43]Maeda Y, Lisi TL, Vance CG Release of GABA and activation of GABA (A) in t he spinal cord mediates the effects of TENS in rats [J].Brain Res, 2007,1136(1):43-50.
[44]Rodicio MC, Verona VC, Anton BI, et al. Coloalization of dopamine and GABA in spinal cord neurons in the sea lamprey [J]. Brain research bulletin, 2008,76:45-49.
[45]King BS, Rampil IJ. Anesthetic depression of spinal motor neurons may contribute to lack of movement in response to noxious stimuli [J]. Anesthesiology,1994,81:1484.
[46]Zhou HH, Turndorf H. Hyper- and hypoventilation affects spinal motor neuron excitability during isoflurane anesthesia [J]. Anestha-Analg,1998,87:407.
[1]林春水,于冬男,古妙宁,等.异丙酚靶控输注对健康志愿者脑葡萄糖代谢的影响[J].中华麻醉学杂志.2004,24(9)：645-648.
[2]Minoru M, Kurumi S, Shiro H, et al. GABA receptor activation in the lumbosacral spinal cord decreases detrusor overactivity in spinal cord injured rats[J].The journal of urology,2008,179(5):1178-1183.
[3]Xu AJ, Duan SM, Zeng YM. Effect of intrathecal NMDA and AMPA receptors agonists or antagonists on antinociception of propofol[J]. Acta Pharmacol Sin, 2004,25:9-14.
[4]Rampil, IJ, Mason. Anesthetic potency (MAC) is independent of forebrain structures in the rat[J]. Anesthesiology,1993,78:707-712.
[5]Nicoll RA, Madison DV. General anesthetics hyperpolarize neurons in the vertebrate central nervous system[J]. Science Magazine,1982,217:1055-1057.
[6]Hesdley PM, Parsons, West DC. Opioid receptor-mediated effects on spinal responses to controlled noxious[J]. Fine afferent nerve fibres and pain, 1987,25:620-624.
[7]Rampil IJ, King BS, Volatile anesthetics depress motor neurons[J]. Anesthesiology, 1996,85:129.
[8]Zhou HH, Turndorf H. Hyper- and hypoventilation affects spinal motor neuron excitability during isoflurane anesthesia[J]. Anestha-Analg,1998,87:407.
[9]Sudo M, Sudo S, Chen XG, et al. Thiopental directly depresses lumbar dorsal horn neuronal responses to noxious mechanical stimulation in goats [J]. Acta Anaesthesiol Scand,2001,45:823-829.
[10]Yamamori Y, Kishikawa K,Collins JG. Halothane effects on low-threshold receptive field size of rat spinal dorsal horn neuron appear to be independent of supraspinal modulatory systems [J]. Brain Res,1995;702:162-168.
[11]每晓鹏,王智明,张惠,等。氯胺酮对大鼠脊髓背角胶状质内对突触前神经递质释放的影响[J].神经解剖学杂志.2007,23(3)：239-244.
[12]Jurna L. Depression of nociceptive sensory activity in the rat spinal cord due to the intrathecal administration of drugs:effect of diazepam [J]. Neurosurgery, 1984,15(6):917-20.
[13]Hanzawa, Shinji. Midazolam presynaptically inhibits excitatory synaptic transmission in the superficial dorsal horn of the rat spinal cord [J]. Japanese Journal of Anesthesiology,1999,48(5):474-480.
[14]Cheng G, Kendig JJ. Enflurane decreases glutamate neurotransmission to spinal cord motor neurons by both presynaptic and postsynaptic actions[J]. Anesth Analg,2003,96:1354～1359.
[15]Wong SM, Fong E, Tauck DL, et al. Ethanol as a general anesthetic:actions in spinal cord [J]. Eur J Pharmacol,1997,329:121～127.
[16]Wong SM, Cheng G, Homanics GE, et al. Enflurane actionson spinal cords from mice that lack the beta3 subunit of the GABA(A) receptor [J]. Anesthesiology,2001,95:154～164.
[17]Bohlhalter SM, Hanns M, Jean MF, Inhibitory neurotransmission in rat spinal cord:co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining [J].Brain reseatch,1994,642(11):59-69.
[18]Dong XP, Xu TL. The actions of propofol on gammaaminobutyric acid2A and glycine receptors in acutely dissociated spinal dorsal horn neurons of the rat[J]. Anesth Analg,2002,95:907～914.
[19]Nadeson R, Goodchild CS.Antinociceptive Properties of Propofol: Involvement of Spinal Cord y-Aminobutyric Acid A Receptors [J]. Journal of Pharmacology and Experimental,1997,21(5):210-221.
[20]Sonner JM, Antognini JF, Dutton RC, et al. Inhaled anesthetics and immobility:mechanisms, mysteries, and minimum alveolar anesthetic concentration[J]. Anesth Analg,2003,97:719～740.
[21]Savola MK, Woodley SJ, Kendig JJ. Isoflurane depresses both glutamate- and peptide-mediated slow synaptic transmission in neonatal rat spinal cord[J]. ACAD Sci,1991,625:281-2.
[22]Takenoshita M,Steinbach JH.Halothane blocks low-voltage-activated calcium current in rate sensory neurons[J].Journal of Neuroscience,1991,11:1404-1412.
[23]Wang MY, Rampil IJ, Kendig JJ. Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors[J]. J Pharmacol Exp Ther,1999, 290:362-367.
[24]Ren SL, Wang MY. Supressive effects of propofol on neonate rat spinal cord motoneurons in vitro[J]. Soc Neurosci Abstr,2002,28:446.6.
[25]Asai T, Kusudo K, Ikeda H, et al. Effects of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices:evidence for a presynaptic action[J]. Eur J Neurosci,2002,15:1278～1290.
[26]麻海春,冯春生,曾维安,等.电压依赖性K+通道在大鼠腺苷脑和脊髓镇痛及氟烷麻醉中的中用[J].中华麻醉学杂志,2007,27(4)：373-375.