脑摄取平衡时伤害性刺激条件下丙泊酚在犬脑各区域的分布
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摘要
丙泊酚(Propofol)是目前临床最常用的静脉全麻药,具有起效快、持续时间短、苏醒迅速完全、手术无记忆等优点,被广泛应用于临床麻醉和ICU镇静。脑是静脉麻醉药的效应器官,丙泊酚脑摄取及分布研究是了解其麻醉作用机制的一个重要方面,尽管前期已做了一些工作并取得进展,但仍有待进一步研究。
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具有选择性。近年来对丙泊酚中枢作用的研究已经取得一些进展,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,主要可能是作用于GABA受体,GABA受体激活后,可增加脑神经元电导而产生去极化,从而产生全麻作用。但上述成果仍不能阐明丙泊酚的全麻作用机制。
Upton于1988年提出了脑摄取的概念及其基本研究方法—质量平衡法则(mass balance principles),即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。上世纪90年代后期有不少学者应用质量平衡法则开始了丙泊酚脑摄取的研究,主要利用色谱分析技术测量脑循环动、静脉血中丙泊酚药物浓度变化来计算丙泊酚脑摄取情况。研究表明丙泊酚在脑内基本不代谢、其脑摄取平衡滞后于血药浓度的平衡,认为丙泊酚血药浓度不能反映麻醉深度、丙泊酚全脑摄取量和全脑浓度与麻醉深度存在相关性。基于质量平衡法则的丙泊酚脑摄取研究揭示了丙泊酚在脑内药理学过程的部分规律,指导了以效应室为目标的丙泊酚TCI,取得了良好的临床效果。但脑是一个功能合胞体,不同区域脑组织的解剖和功能有显著不同,不同的脑功能区域对丙泊酚的摄取规律也可能不同。由于实验方法和技术的限制,基于质量平衡法则的脑摄取研究,只能计算药物的脑摄取,所反映的是药物的全脑摄取而不能反映药物在不同脑区的实际摄取和分布特征。
为克服以前丙泊酚脑摄取研究的不足,Shyr和Larsson分别探索了新的脑摄取研究方法,即基于解剖脑组织的直接法脑摄取研究：通过解剖丙泊酚麻醉状态下的动物脑组织并直接测量脑组织丙泊酚浓度,可以直观的获得不同部位脑组织对丙泊酚摄取规律及其它脑内药理学特征。直接法脑摄取研究的优势在于：能直接了解丙泊酚实际脑浓度,可以反映在达到某个麻醉状态即刻丙泊酚脑摄取的规律和丙泊酚脑内分布情况,能进一步明确脑摄取规律及脑浓度变化特征。Shyr观察到,丙泊酚60 mg／(kg·h)恒速输注30min,丙泊酚在大鼠脑皮层、海马、纹状体、间脑、中脑、脑桥、小脑、延髓的分布均衡。而Larsson观察了大鼠皮层、中脑(含脑干)和小脑三个区域,丙泊酚注射速度分别为2.5、5、10 mg/(kg-h),287和776天鼠达到麻醉状态即刻中脑丙泊酚浓度高于其它脑组织；注射速度为20 mg/(kg-h)组和23天鼠龄组(以上述四种速度)达到麻醉状态即刻,三区域脑组织丙泊酚均呈均衡分布。二者的结论不尽相同,这可能与注射速度、鼠龄和所观察区域的多少有关；恒速输注速度快、鼠龄小,丙泊酚脑内分布可能容易达到均衡。
丙泊酚脑摄取和分布规律还可能受到注射方式、脑摄取状态、动物物种差异等因素的影响。以前的实验多采用SD大鼠为实验动物,由于鼠脑结构和功能与人脑差别较大,难以将实验结果进行推广,因此有必要对更高等的动物加以研究。犬脑为沟回脑,脑功能分区和结构与人脑相似,脑体积和质量较大,解剖标志明显,利于实验操作。我们前期研究发现,单次静脉注射达到不同的麻醉深度时,犬不同脑组织区域丙泊酚的分布不同：浅麻醉状态时桥脑丙泊酚浓度最高,深麻醉状态时丘脑最高,海马最低。丙泊酚70mg/(kg.h)恒速静脉输注30min,犬脑循环动、静脉血药浓度达平衡状态,此时丙泊酚脑摄取达动态平衡,除背侧丘脑丙泊酚浓度较高外,在其他区域组织分布均衡。最近研究发现,丙泊酚70mg/(kg.h)恒速静脉输注50min,丙泊酚脑摄取才处于稳定平衡状态,在犬脑各区域(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑、延髓、颈髓)呈均衡分布。但在手术刺激状态,伤害性刺激是否改变脑摄取的平衡?是否引起丙泊酚在不同脑区分布的改变?有待研究。
本研究采用高效液相色谱紫外法(High-pressure liquid chromatography ultra-violet spectroscopy, HPLC-UV),测量犬脑组织不同区域(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑、延髓、颈髓)丙泊酚浓度,探讨脑摄取平衡时伤害性刺激条件下丙泊酚在犬脑不同区域的分布特征和规律。
材料和方法
1实验动物准备与分组：
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。
1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50min。
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50min。当丙泊酚输注50min时止血钳上三齿钳夹狗尾正中lmin。
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：
S组输注51min时、C组输注50min时,于右侧颈内动、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,4℃冰箱中保存。取血后立即断头法处死实验犬,无菌条件下去除颅盖等组织,专人解剖获取背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织,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30min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。
脑组织标本精确称量(g)后移于匀浆器中,每克脑组织中加入乙腈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,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样本均数比较的t检验；组间颈内动脉和静脉血浆丙泊酚浓度比较采用两样本均数比较的t检验。组内不同标本中丙泊酚浓度比较采用重复测量设计资料的方差分析,多重比较采用LSD检验。组间相同部位标本中丙泊酚浓度比较采用两样本均数比较的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：79.46±1.29次·min-1、88.19±1.21mmHg和35.74±1.92mmHg。S组刺激前后PR分别为：82.59±1.79次min-1和95.09±1.85次min-1,刺激后PR明显高于刺激前(t=21.226,P=0.000)；S组刺激前后MAP分别为：89.48±1.18mmHg和102.34±1.64mmHg,刺激后MAP明显高于刺激前(t=14.865,P=0.000)；S组刺激前后PETCO2分别为35.17±1.21mmHg和35.22±1.35mmHg,二者差异无统计学意义(t=0.431,P=0.761)。
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6.17±1.00μg/ml和6.16±0.96μg/ml,二者差异无统计学意义(t=0.252,P=0.811)。
S组颈内动脉和颈内静脉血浆丙泊酚浓度分别为6.16±1.04μg/ml和6.16±0.49μg/ml,二者差异无统计学意义(t=0.002,P=0.999)。
C组和S组比较,颈内动脉血浆丙泊酚浓度差异无统计学意义(t=-0.020,P=0.984)；颈内静脉血浆丙泊酚浓度差异无统计学意义(t=0.000,P=1.000)。
3丙泊酚脑组织浓度：
C组犬背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织丙泊酚浓度(μg·g-1)分别为6.12±0.50、6.08±0.67、6.08±0.89、6.21±0.73、6.12±0.51、6.08±0.31、6.05±0.63、6.12±0.70、6.09±0.58、6.06±0.97、6.08±1.04、6.03±0.99、6.11±0.75,各区域脑组织丙泊酚浓度差异无统计学意义(F=0.037,P=0.948)。
S组犬背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织丙泊酚浓度(μg·g-1)分别为：8.99±0.77、6.13±1.10、6.12±1.10、6.11±0.94、9.13±0.53、6.11±0.99、6.11±0.93、6.10±0.90、6.10±0.94、6.13±0.96、6.11±1.04、6.10±0.93、6.11±0.96,S组各区域脑组织丙泊酚浓度差异有统计学意义(F=17.207,P=0.000),其中背侧丘脑和底丘脑丙泊酚浓度分别为(8.99±0.77)μg／g和(9.13±0.53)μg／g,明显高于其他脑区浓度,差异有统计学意义(P<0.05),背侧丘脑和底丘脑丙泊酚浓度差异无统计学意义(P=0.643)；其余不同脑区丙泊酚浓度差异无统计学意义(P>0.05)。
S组背侧丘脑和底丘脑丙泊酚浓度明显高于C组相应脑区丙泊酚浓度,差异有统计学意义(P<0.05),其他相同区域组间比较差异无统计学意义(P>0.05)。
刺激因素及脑组织区域因素之间存在交互效应(F=10.575,P=0.000),脑摄取平衡时伤害性刺激条件下丙泊酚在犬脑背侧丘脑及底丘脑分布浓度最高。
结论
1.丙泊酚恒速70mg·kg-1·h-1恒速静脉输注50分钟,颈内动、静脉血药浓度和丙泊酚脑摄取达到稳定的平衡状态,丙泊酚在各区域脑组织(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑)的分布均衡。2.脑摄取平衡状态时给予伤害性刺激,颈内动、静脉丙泊酚浓度较刺激前和未刺激组均无明显变化,脑摄取仍处于稳定的平衡状态；而背侧丘脑和底丘脑丙泊酚浓度明显高于其他区域,丙泊酚在其余各解剖区域的分布均衡。
Owing to the advantage of quick effect、short duration、prompt revival and surgery-free memory, Propofol (2,6-diisopropylphenol) has been widely used in general anesthesia induction and maintenance and used as a sedative agent in the intensive care unit (ICU). Many researches have been done to investigate the anesthetic mechanism of propofol, but it has not been made out very clearly today. As we know, brain is the effective organ of intravenous anesthetics, the research of cerebeal uptake and distribution of popofol is favourable to explore the mechanism of general anesthesia of popofol.
The anatomic structure and distribution of central nervous systems have a certain degree of specificity, so that the anesthetic agents to play its role in the pharmacology of narcotic site also selective. The central GABAA receptor is the major inhibitory receptor in the central nervous system. It scatters in the brain, but the distribution is disequilibrium in different brain tissues and it is more intensive in some cerebral tissues. A number of studys showed that the central GABAA receptor represents an important target in mediating the anesthetic mechanism of propofol. Synaptic theory is one of the most important theories of general mechanism.The doctrine suppose that general anesthesia drugs may target on the synaptic transmission, the main function of the propofol may be on GABA receptors. GABA receptors activated can increase nervous conductance and depolarization, resulting in the anesthesia process. Basing on these researches, it can be assumed that propofol concentrations are discordant in various cerebral tissues. Therefore, the cerebral uptake and regional distribution of propofol must be investigated for the understanding of its anesthetic mechanism.
1、In 1988, Upton put forward the concept of cerebral uptake and the method of mass balancing principles. By measuring the propofol concentrations in arterial and venous blood of cerebral circulation, in addition cerebral blood flow, we can calculate the feature of the cerebral uptake of propofol indirectly, but the character of regional distribution of propofol in the brain can not be revealed objectively yet by this method. To improve the research of cerebral uptake of propofol, the propofol concentrations in different parts of the brain must be detected directly by anatomical methods. Shyr and Larsson had compared the propofol distribution of brain tissues in the Sprague Dawley rat respectively. Shyr discovered that the cerebral uptake of propofol was equilibrium in different brain tissues,but Larsson discovered that the midbrain was different to the other brain function area in intaking propofol. It may be injected factor, state of brain uptake, and species of experimental animal that influence the cerebral uptake and distribution of propofol. The previous experiments used the SD rat as the experimental animal. Due to the significant differences between the mouse brain structure and function and the human brain's, it is difficult to generalize the experimental results. It is necessary to further study with a more high-grade animal. The function district and the structure of dog brain are similar with that of the human brain, and the volume and the quality of dog brain are bigger than the mouse brain's, furthermore, there has obvious anatomical landmark so as to get the obvious regional tissues in dog brain. We had measured the propofol concentrations in different cerebral tissues under different depth of anesthesia at a single bolus in dogs. Experimental results shown that the propofol concentration was highest in pons when the animal's eyes closed without stimulation and in thalamus when the eyelid reflex disappeared respectively, and lowest in hippocampus under the both anesthesia conditions.Under different depth of anesthesia at a single bolus intravenous injection of propofol, the difference of propofol concentrations between internal carotid artery and jugular vein was significant and showed that the cerebral uptake of propofol was underway. The late study indicated that at 30 min a constant rate of 70mg/(kg·h) intravenous injection of propofol, propofol was distributed dynamic evenly among regional cerebral tissues in dogs, but the thalamus constains high propofol concentration; at 50 min cerebral uptake of propofol achieved stable equilibrium state, propofol was distributed evenly in the cerebral tissues in dogs.
The chief aim of this study is to mesure the propofol concentrations of different cerebral tissues bying HPLC-UV and to investigate the cerebral uptake and regional distribution of propofol under the circumstance of noxious stimulation at cerebral uptake quilibrium of propofol in dogs.
Material and methods
12 healthy male dogs aged 12-18 months were divided randomly into two groups (group S and C). All the experiment were scheduled during the day and fasting in diet for 12-hour were 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 70mg/(kg·h) to maintain anesthesia.
When the infusion of propofol was at the 50th min,animals of group S were given stimulation to the end of its tail by hemostat for lmin. Animals of group C were given no stimulation.The blood samples were taken from the right internal carotid and internal jugular vein at the 51th min in group S and at the 50th min in group C. Then the animal was scarificed immediately by decapitation. The dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrai、pons、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,10×4.6mmID 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). 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.
2.The propfol concentration in blood plasma:
The concentration of propofol in internal carotid artery and internal jugular vein blood plasma were 6.16±1.04、6.16±0.49μg/ml (t=0.002,P=0.999) in group S and 6.17±1.00、6.16±0.99μg/ml(t=0.252,P=0.811)in group C respectively, the differences between them were not statistically significant.
3. The propofol concentration in brain tissues:
In group C, the propofol concentrations of the dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons were 6.12±0.50、6.08±0.67、6.08±0.89、6.21±0.73、6.12±0.51、6.08±0.31、6.05±0.63、6.12±0.70、6.09±0.58、6.06±0.97、6.08±1.04、6.03±0.99、6.11±0.75(μg/g) respectively(F=0.037, P=0.948) and no significant differences.
In group S, the propofol concentrations of dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons were 8.99±0.77、6.13±1.10、6.12±1.10、6.11±0.94、9.13±0.53、6.11±0.99、6.11±0.93、6.10±0.90、6.10±0.94、6.13±0.96、6.11±1.04、6.10±0.93、6.11±0.96 (μg·g-1) respectively (F=17.207,P=0.000). The propofol concentration in the dorsal thalamus (8.99±0.77μg·g-1) and subthalamus (9.13±0.53ug/g) was higher than these any other brain tissues'(P<0.05).
The propofol concentrations were no significant difference between group S and C (P> 0.05).The brain tissues concentrations of propofol exist interaction with noxious stimulus (F=10.575, P=0.000).The concentration of propofol in dorsal thalamus and subthalamus were highest under the condition of balance of the brain uptake and noxious stimulus.
Conclusions
1 At 50 min a constant rate of 70mg/(kg-h) intravenous injection of propofol, there is no significant differences in propofol concentration between jugular venous blood and carotid arterial blood and show whole brain uptake of propofol achieve a equilibrium state. Propofol is distributed evenly among all the region of brain tissues.
2 Under the circumstance of noxious stimulation, the propofol concentration in dorsal thalamus and subthalamus are higher than that in other cerebral regions (epithalamus、metathalamus、hypothalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons),which is in equilibrious distribution of propofol in the later.
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具有选择性。近年来对丙泊酚中枢作用的研究已经取得一些进展,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,主要可能是作用于GABA受体,GABA受体激活后,可增加脑神经元电导而产生去极化,从而产生全麻作用。但上述成果仍不能阐明丙泊酚的全麻作用机制。
Upton于1988年提出了脑摄取的概念及其基本研究方法—质量平衡法则(mass balance principles),即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。上世纪90年代后期有不少学者应用质量平衡法则开始了丙泊酚脑摄取的研究,主要利用色谱分析技术测量脑循环动、静脉血中丙泊酚药物浓度变化来计算丙泊酚脑摄取情况。研究表明丙泊酚在脑内基本不代谢、其脑摄取平衡滞后于血药浓度的平衡,认为丙泊酚血药浓度不能反映麻醉深度、丙泊酚全脑摄取量和全脑浓度与麻醉深度存在相关性。基于质量平衡法则的丙泊酚脑摄取研究揭示了丙泊酚在脑内药理学过程的部分规律,指导了以效应室为目标的丙泊酚TCI,取得了良好的临床效果。但脑是一个功能合胞体,不同区域脑组织的解剖和功能有显著不同,不同的脑功能区域对丙泊酚的摄取规律也可能不同。由于实验方法和技术的限制,基于质量平衡法则的脑摄取研究,只能计算药物的脑摄取,所反映的是药物的全脑摄取而不能反映药物在不同脑区的实际摄取和分布特征。
为克服以前丙泊酚脑摄取研究的不足,Shyr和Larsson分别探索了新的脑摄取研究方法,即基于解剖脑组织的直接法脑摄取研究：通过解剖丙泊酚麻醉状态下的动物脑组织并直接测量脑组织丙泊酚浓度,可以直观的获得不同部位脑组织对丙泊酚摄取规律及其它脑内药理学特征。直接法脑摄取研究的优势在于：能直接了解丙泊酚实际脑浓度,可以反映在达到某个麻醉状态即刻丙泊酚脑摄取的规律和丙泊酚脑内分布情况,能进一步明确脑摄取规律及脑浓度变化特征。Shyr观察到,丙泊酚60 mg／(kg·h)恒速输注30min,丙泊酚在大鼠脑皮层、海马、纹状体、间脑、中脑、脑桥、小脑、延髓的分布均衡。而Larsson观察了大鼠皮层、中脑(含脑干)和小脑三个区域,丙泊酚注射速度分别为2.5、5、10 mg/(kg-h),287和776天鼠达到麻醉状态即刻中脑丙泊酚浓度高于其它脑组织；注射速度为20 mg/(kg-h)组和23天鼠龄组(以上述四种速度)达到麻醉状态即刻,三区域脑组织丙泊酚均呈均衡分布。二者的结论不尽相同,这可能与注射速度、鼠龄和所观察区域的多少有关；恒速输注速度快、鼠龄小,丙泊酚脑内分布可能容易达到均衡。
丙泊酚脑摄取和分布规律还可能受到注射方式、脑摄取状态、动物物种差异等因素的影响。以前的实验多采用SD大鼠为实验动物,由于鼠脑结构和功能与人脑差别较大,难以将实验结果进行推广,因此有必要对更高等的动物加以研究。犬脑为沟回脑,脑功能分区和结构与人脑相似,脑体积和质量较大,解剖标志明显,利于实验操作。我们前期研究发现,单次静脉注射达到不同的麻醉深度时,犬不同脑组织区域丙泊酚的分布不同：浅麻醉状态时桥脑丙泊酚浓度最高,深麻醉状态时丘脑最高,海马最低。丙泊酚70mg/(kg.h)恒速静脉输注30min,犬脑循环动、静脉血药浓度达平衡状态,此时丙泊酚脑摄取达动态平衡,除背侧丘脑丙泊酚浓度较高外,在其他区域组织分布均衡。最近研究发现,丙泊酚70mg/(kg.h)恒速静脉输注50min,丙泊酚脑摄取才处于稳定平衡状态,在犬脑各区域(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑、延髓、颈髓)呈均衡分布。但在手术刺激状态,伤害性刺激是否改变脑摄取的平衡?是否引起丙泊酚在不同脑区分布的改变?有待研究。
本研究采用高效液相色谱紫外法(High-pressure liquid chromatography ultra-violet spectroscopy, HPLC-UV),测量犬脑组织不同区域(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑、延髓、颈髓)丙泊酚浓度,探讨脑摄取平衡时伤害性刺激条件下丙泊酚在犬脑不同区域的分布特征和规律。
材料和方法
1实验动物准备与分组：
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。
1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50min。
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50min。当丙泊酚输注50min时止血钳上三齿钳夹狗尾正中lmin。
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：
S组输注51min时、C组输注50min时,于右侧颈内动、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,4℃冰箱中保存。取血后立即断头法处死实验犬,无菌条件下去除颅盖等组织,专人解剖获取背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织,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30min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。
脑组织标本精确称量(g)后移于匀浆器中,每克脑组织中加入乙腈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,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样本均数比较的t检验；组间颈内动脉和静脉血浆丙泊酚浓度比较采用两样本均数比较的t检验。组内不同标本中丙泊酚浓度比较采用重复测量设计资料的方差分析,多重比较采用LSD检验。组间相同部位标本中丙泊酚浓度比较采用两样本均数比较的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：79.46±1.29次·min-1、88.19±1.21mmHg和35.74±1.92mmHg。S组刺激前后PR分别为：82.59±1.79次min-1和95.09±1.85次min-1,刺激后PR明显高于刺激前(t=21.226,P=0.000)；S组刺激前后MAP分别为：89.48±1.18mmHg和102.34±1.64mmHg,刺激后MAP明显高于刺激前(t=14.865,P=0.000)；S组刺激前后PETCO2分别为35.17±1.21mmHg和35.22±1.35mmHg,二者差异无统计学意义(t=0.431,P=0.761)。
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6.17±1.00μg/ml和6.16±0.96μg/ml,二者差异无统计学意义(t=0.252,P=0.811)。
S组颈内动脉和颈内静脉血浆丙泊酚浓度分别为6.16±1.04μg/ml和6.16±0.49μg/ml,二者差异无统计学意义(t=0.002,P=0.999)。
C组和S组比较,颈内动脉血浆丙泊酚浓度差异无统计学意义(t=-0.020,P=0.984)；颈内静脉血浆丙泊酚浓度差异无统计学意义(t=0.000,P=1.000)。
3丙泊酚脑组织浓度：
C组犬背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织丙泊酚浓度(μg·g-1)分别为6.12±0.50、6.08±0.67、6.08±0.89、6.21±0.73、6.12±0.51、6.08±0.31、6.05±0.63、6.12±0.70、6.09±0.58、6.06±0.97、6.08±1.04、6.03±0.99、6.11±0.75,各区域脑组织丙泊酚浓度差异无统计学意义(F=0.037,P=0.948)。
S组犬背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑组织丙泊酚浓度(μg·g-1)分别为：8.99±0.77、6.13±1.10、6.12±1.10、6.11±0.94、9.13±0.53、6.11±0.99、6.11±0.93、6.10±0.90、6.10±0.94、6.13±0.96、6.11±1.04、6.10±0.93、6.11±0.96,S组各区域脑组织丙泊酚浓度差异有统计学意义(F=17.207,P=0.000),其中背侧丘脑和底丘脑丙泊酚浓度分别为(8.99±0.77)μg／g和(9.13±0.53)μg／g,明显高于其他脑区浓度,差异有统计学意义(P<0.05),背侧丘脑和底丘脑丙泊酚浓度差异无统计学意义(P=0.643)；其余不同脑区丙泊酚浓度差异无统计学意义(P>0.05)。
S组背侧丘脑和底丘脑丙泊酚浓度明显高于C组相应脑区丙泊酚浓度,差异有统计学意义(P<0.05),其他相同区域组间比较差异无统计学意义(P>0.05)。
刺激因素及脑组织区域因素之间存在交互效应(F=10.575,P=0.000),脑摄取平衡时伤害性刺激条件下丙泊酚在犬脑背侧丘脑及底丘脑分布浓度最高。
结论
1.丙泊酚恒速70mg·kg-1·h-1恒速静脉输注50分钟,颈内动、静脉血药浓度和丙泊酚脑摄取达到稳定的平衡状态,丙泊酚在各区域脑组织(背侧丘脑、上丘脑、后丘脑、下丘脑、底丘脑、额叶、顶叶、颞叶、海马、扣带回、小脑、中脑、桥脑)的分布均衡。2.脑摄取平衡状态时给予伤害性刺激,颈内动、静脉丙泊酚浓度较刺激前和未刺激组均无明显变化,脑摄取仍处于稳定的平衡状态；而背侧丘脑和底丘脑丙泊酚浓度明显高于其他区域,丙泊酚在其余各解剖区域的分布均衡。
Owing to the advantage of quick effect、short duration、prompt revival and surgery-free memory, Propofol (2,6-diisopropylphenol) has been widely used in general anesthesia induction and maintenance and used as a sedative agent in the intensive care unit (ICU). Many researches have been done to investigate the anesthetic mechanism of propofol, but it has not been made out very clearly today. As we know, brain is the effective organ of intravenous anesthetics, the research of cerebeal uptake and distribution of popofol is favourable to explore the mechanism of general anesthesia of popofol.
The anatomic structure and distribution of central nervous systems have a certain degree of specificity, so that the anesthetic agents to play its role in the pharmacology of narcotic site also selective. The central GABAA receptor is the major inhibitory receptor in the central nervous system. It scatters in the brain, but the distribution is disequilibrium in different brain tissues and it is more intensive in some cerebral tissues. A number of studys showed that the central GABAA receptor represents an important target in mediating the anesthetic mechanism of propofol. Synaptic theory is one of the most important theories of general mechanism.The doctrine suppose that general anesthesia drugs may target on the synaptic transmission, the main function of the propofol may be on GABA receptors. GABA receptors activated can increase nervous conductance and depolarization, resulting in the anesthesia process. Basing on these researches, it can be assumed that propofol concentrations are discordant in various cerebral tissues. Therefore, the cerebral uptake and regional distribution of propofol must be investigated for the understanding of its anesthetic mechanism.
1、In 1988, Upton put forward the concept of cerebral uptake and the method of mass balancing principles. By measuring the propofol concentrations in arterial and venous blood of cerebral circulation, in addition cerebral blood flow, we can calculate the feature of the cerebral uptake of propofol indirectly, but the character of regional distribution of propofol in the brain can not be revealed objectively yet by this method. To improve the research of cerebral uptake of propofol, the propofol concentrations in different parts of the brain must be detected directly by anatomical methods. Shyr and Larsson had compared the propofol distribution of brain tissues in the Sprague Dawley rat respectively. Shyr discovered that the cerebral uptake of propofol was equilibrium in different brain tissues,but Larsson discovered that the midbrain was different to the other brain function area in intaking propofol. It may be injected factor, state of brain uptake, and species of experimental animal that influence the cerebral uptake and distribution of propofol. The previous experiments used the SD rat as the experimental animal. Due to the significant differences between the mouse brain structure and function and the human brain's, it is difficult to generalize the experimental results. It is necessary to further study with a more high-grade animal. The function district and the structure of dog brain are similar with that of the human brain, and the volume and the quality of dog brain are bigger than the mouse brain's, furthermore, there has obvious anatomical landmark so as to get the obvious regional tissues in dog brain. We had measured the propofol concentrations in different cerebral tissues under different depth of anesthesia at a single bolus in dogs. Experimental results shown that the propofol concentration was highest in pons when the animal's eyes closed without stimulation and in thalamus when the eyelid reflex disappeared respectively, and lowest in hippocampus under the both anesthesia conditions.Under different depth of anesthesia at a single bolus intravenous injection of propofol, the difference of propofol concentrations between internal carotid artery and jugular vein was significant and showed that the cerebral uptake of propofol was underway. The late study indicated that at 30 min a constant rate of 70mg/(kg·h) intravenous injection of propofol, propofol was distributed dynamic evenly among regional cerebral tissues in dogs, but the thalamus constains high propofol concentration; at 50 min cerebral uptake of propofol achieved stable equilibrium state, propofol was distributed evenly in the cerebral tissues in dogs.
The chief aim of this study is to mesure the propofol concentrations of different cerebral tissues bying HPLC-UV and to investigate the cerebral uptake and regional distribution of propofol under the circumstance of noxious stimulation at cerebral uptake quilibrium of propofol in dogs.
Material and methods
12 healthy male dogs aged 12-18 months were divided randomly into two groups (group S and C). All the experiment were scheduled during the day and fasting in diet for 12-hour were 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 70mg/(kg·h) to maintain anesthesia.
When the infusion of propofol was at the 50th min,animals of group S were given stimulation to the end of its tail by hemostat for lmin. Animals of group C were given no stimulation.The blood samples were taken from the right internal carotid and internal jugular vein at the 51th min in group S and at the 50th min in group C. Then the animal was scarificed immediately by decapitation. The dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrai、pons、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,10×4.6mmID 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). 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.
2.The propfol concentration in blood plasma:
The concentration of propofol in internal carotid artery and internal jugular vein blood plasma were 6.16±1.04、6.16±0.49μg/ml (t=0.002,P=0.999) in group S and 6.17±1.00、6.16±0.99μg/ml(t=0.252,P=0.811)in group C respectively, the differences between them were not statistically significant.
3. The propofol concentration in brain tissues:
In group C, the propofol concentrations of the dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons were 6.12±0.50、6.08±0.67、6.08±0.89、6.21±0.73、6.12±0.51、6.08±0.31、6.05±0.63、6.12±0.70、6.09±0.58、6.06±0.97、6.08±1.04、6.03±0.99、6.11±0.75(μg/g) respectively(F=0.037, P=0.948) and no significant differences.
In group S, the propofol concentrations of dorsal thalamus、epithalamus、metathalamus、hypothalamus、subthalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons were 8.99±0.77、6.13±1.10、6.12±1.10、6.11±0.94、9.13±0.53、6.11±0.99、6.11±0.93、6.10±0.90、6.10±0.94、6.13±0.96、6.11±1.04、6.10±0.93、6.11±0.96 (μg·g-1) respectively (F=17.207,P=0.000). The propofol concentration in the dorsal thalamus (8.99±0.77μg·g-1) and subthalamus (9.13±0.53ug/g) was higher than these any other brain tissues'(P<0.05).
The propofol concentrations were no significant difference between group S and C (P> 0.05).The brain tissues concentrations of propofol exist interaction with noxious stimulus (F=10.575, P=0.000).The concentration of propofol in dorsal thalamus and subthalamus were highest under the condition of balance of the brain uptake and noxious stimulus.
Conclusions
1 At 50 min a constant rate of 70mg/(kg-h) intravenous injection of propofol, there is no significant differences in propofol concentration between jugular venous blood and carotid arterial blood and show whole brain uptake of propofol achieve a equilibrium state. Propofol is distributed evenly among all the region of brain tissues.
2 Under the circumstance of noxious stimulation, the propofol concentration in dorsal thalamus and subthalamus are higher than that in other cerebral regions (epithalamus、metathalamus、hypothalamus、frontal lobe、parietal lobe、temporal lobe、hippocampus、cingulate gyrus、cerebellum、midbrain、pons),which is in equilibrious distribution of propofol in the later.
引文
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