制首乌对叠氮钠脑内灌流大鼠的脑保护机制研究
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摘要
1.研究目的与依据
老年性痴呆(Alzheimer's Disease, AD)是严重危害老年人健康的疾病之一。脑内胆碱能神经退行性变导致AD认知功能缺损的观点,已被广泛接受。乙酰胆碱(Acetylcholine, Ach)是胆碱能神经传导的内源性化学递质,Ach脑内水平的平衡调节对维持学习、记忆、注意力等脑高级功能的正常运作至关重要。脑内微透析(Microdialysis)技术是一种在麻醉或清醒自由活动动物脑内取样的新手段,它的出现为脑内细胞外环境进行在体采样和动态监测提供了方便。
近年来,线粒体氧化磷酸化异常在AD发病中的作用引起人们的重视。叠氮钠(NaN_3)作为细胞色素C氧化酶抑制剂,能引起线粒体氧化磷酸化异常。研究表明,脑外给予NaN_3可导致动物学习记忆能力下降、胆碱能神经功能下降等与AD的病理改变相似的病变。本研究拟采用脑内灌流0.2%叠氮钠120min的方式造成胆碱能神经功能障碍的模型,为寻找新的治疗痴呆的药物提供可参考的方法。
何首乌是传统的常用的补益中药,有滋补肝肾,延年益寿,健脑调心的功效。已有的研究证实它具有改善学习记忆功能等药理作用,提示何首乌可能对脑有保护作用。本研究中选用已证实有效的剂量的制首乌水煎液进行研究。旨在探讨制首乌对叠氮钠灌流所致的大鼠脑内乙酰胆碱水平下降的调节作用及其机理。为何首乌作为脑保护剂治疗老年人常见的退行性疾病所致的痴呆提供实验依据。
2.研究方法
(1) 微透析技术动态监测脑内Ach/Ch水平变化:微透析技术是利用小分子物质能通过半透膜顺浓度梯度进行扩散的原理为基础进行脑内采样的技术。本研究采用此方法实现对清醒自由活动大鼠脑内细胞外Ach/Ch水
1. PurposeAlzheimer's Disease (AD) is one of the most severe diseases that damages the health of the senior citizen. It has been widely accepted that retrograde cholinergic nerve causes cognitive defect in AD patients. Acetylcholine (Ach) is the transmitter of cholinergic nerve, and the maintenance of balanced level of Ach in brain plays a important role in keeping the normal advanced function of brain, such as learning, memory and attention. Microdialysis is a new sampling method in brain of anaesthetized or conscious animals, and it facilitates sampling from brain extracellular environment in vivo and dynamiclly detecting.Recently, abnormal oxidative phosphorylation in mitochondria attracted much attention by its role in AD. Sodium azide (NaN_3), as an inhibitor of cytochrome oxidase (COX), can induce the abnormity of oxidative phosphorylation in mitochondria. Animal tests showed that NaN_3 administration outside of brain could reduce the learning and memory abilities, function of cholinergic function as well, which are similar to those of AD. In this study, we manage to made a animal model with reducing cholinergic function by NaN_3 perfusion 120 minutes in brain.Polygonum multiflorum Thumb. is a traditional material medica commonly used to nourish liver and kidney, prolong life, benefit brain and coordinate heart. It has been proved that it has the function of improving learning and memory abilities, which indicated that it may protect brain. In this study, the confirmed
effective dose of decoct of Radix Polygoni Multiflori Preparata (RPMP) was used to find out its function and mechanism on adjusting the Ach level in rat's brain with NaN3 perfusion. It may provide the experimental evidence to support the idea that Polygonum multiflorum Thumb, could be brain protective drug to treat retrogressive disease such as dementia.2. Method2.1 Dynamically inspecting on the changes of Ach/Ch levels in brain with microdialysis: Microdialysis is a kind of sampling technique in brain which based on the principle that small molecule can penetrate semipermeable membranes. In this study, the method was used for dynamically detecting on the changes of Ach/Ch levels in the brain of conscious free moving rats.2.2 Determining the content of Ach and Ch in dialysate with the method of high performance liquid chromatography - Immobilized enzyme reactor -electrochemical detection (HPLC-IMER-ED): Based on the operation manual( MF-9053 ) from BAS company and related literatures, this method was used to determine the content of Ach/Ch which was collected by Microdialysis collection. Methodology test show that it is sensitive and reliable.2.3 Determining the activity of choline-acetyl-transfertase (ChAT) with radio isotope biding method.2.4 Determining the activity of Acetylcholinesterase (AchE) with spectrophotometry2.5 Determining the binding ability of cholinergic M receptor (M-R) and N-methyl-D-aspartic acid receptor (NMDA-R) with radioactive aglucone -receptor biding method2.6 Determining the content of LD with spectrophotometry3. Content3.1 Study on the animal model with reducing cholinergic function by NaN3 perfusion 120 minutes in brain.
3.1.1 Groups and treatments of animalsSD rats were randomly divided into two groups: the control group and the model group.Protocol of Microdialysis: implanted a probe into striatum for perfusion and collected one vial of dialysate per 30 min. The process could be divided into 3 steps for the model rats: pre-perfusion of NaN3, perfusion of NaN3 (2 h) and after NaN3 perfusion (16.5 h, recovery period), while the control group rats were perfused with Ringer solution all along.3.1.2 Index of research? The content of Ach and Ch indialysate. ? decollate rats after microdialysis progress and remove the striatum, then determined the activity of ChAT, AchE, M-R and NMDA-R, the content of lactic acid (LD) as well.3.1.3 Statistical analysisThe data of the two groups were showed with Mean ± Sd, and t-test was used to compare mean value3.1.4 Results(1) The changes of Ach: During the whole observation period, the extracellular Ach level of the control group in striatum stayed stable, with fluctuation occasionally. While the level of the model rats dropped greatly during the period of NaN3 perfusion. The Ach level stopped dropping after NaN3 perfusion, it stayed stable and showed the tendency of increasing slowly from 5.5h later, and at 14.5h in recovery period, the Ach level reached relatively equal to that of control group.(2) The changes of Ch level: The Ch level of the control group stayed stable during the whole period of microdialysis. However, the Ch level of model increased rapidly immediately when NaN3 perfusion starting, reached its peak lh later, then entered the platform phrase after this. After NaN3 perfusion the Ch level began to drop, and stayed stable 4h later.(3) The changes of other indexes: 16.5 h later after NaN3 perfusion, the activity of ChAT and M-R in the model group decreased with no statistical
significance, compared to those of the control. AchE activity and the content of lactic acid of the two groups were very close to each other. The activity of NMDA-R in the model group increased significantly.3.1.5 DiscussionThe extracellular Ach level in the brain can mirror the cholinergic function in brain reliably. The results showed the extracellular Ach level in striatum decreased caused by 0.2% NaN3 perfusion 120min through microdialysis administration, and this changes were reversible with its level return to the normal value 14.5h after NaN3 perfusion. So, the animal model was successfully made.Ch is the material of synthesizing Ach, and its cleavage product as well. This study showed extracellular Ch level increased significantly caused by NaN3. Such changes may relate to the uptake disorder of Ch, which result the decreasing synthesis of Ach in neurons.The study on neurotransmitter can not be limited to the changes itself, but the analysis of its receptor and related enzymes should also be conducted to make clear the changes of transmitter system on a wide scale. The activity of ChAT, AchE and M-R did not showed the significantly changes in this test. The content of LD, having the function of reflecting mitochondria function decreasing, did not change significantly either. Such negative results may relate to the time of sampling: the sampling began at 16.5h after NaN3 perfusion, when these changes had weaken even disappeared.The toxic effects of excitatory amino acid (EAA) induced by super-activation of NMDA-R plays an important role in retrograde neuropathogenesis such as AD. This study showed the activity of NMDA-R increased significantly caused by NaN3 perfusion.3.1.6 Summary(1) In this study, the dynamically changes of extracellular Ach and Ch in striatum during 0.2% NaN3 perfusion 120 min and recovery period were observed in a long period. The results of reversible decreasing of Ach level and increasing Ch level indicated the animal model that imitate the reducing of
cholinergic nerve function was successfully made.(2) The negative results indicated that the damage may have recovered when sampling conducted at 16.5h after NaN3 perfusion.. Based on the results of Ach and Ch, we plan to decollate animals and remove striatum when 3.5 h after NaN3 perfusion in order to observe the changes of relative indexes in the later research..3.2 Study on Protection Mechanism of Radix Polygoni Multiflori Preparata in Rats Brain with Sodium Azide Perfusion3.2.1 Groups and treatmentsSD rats were randomly divided into 5 groups: RPMP group (15g/Kg), Duxil group (lOmg/Kg), RPMP & Duxil group (RPMP 15g/Kg & Duxil lOmg/Kg), Control and Model (distilled water). Followed by the above doses, rats were i.g. for 14 days continuously. On the 13th day, guide tube was implanted into the brain of rat and then the rats were operated with microdialysis the next day.Microdialysis protocol: implanted a probe into striatum for perfusion and collected one vial of dialysate per 30 min. Control was perfused with Ringer solution all along. Process could be divided into 3 steps for the other groups: pre-perfusion of NaN3, perfusion of NaN3 (2 h) and after NaN3 perfusion (3.5 h, recovery period).3.2.2 Index of research? The content of Ach and Ch in dialysate, their area under the content-time curve, extreme values and the extreme time. @ decollate rats after Microdialysis progress and remove the striatum, then determined the activity of ChAT, AchE, M-R and NMDA-R, the content of lactic acid (LD) as well.3.2.3 Statistical analysisIndex of each group was showed with Mean ± Sd. ANOVA was used, and if there is significant difference then use LSD method to compare the average among goups.3.2.4 Results
(1) Changes of Ach: The extracellular Ach level of striatum stayed stable in the control group during the whole observing period but dropped significantly in the model group during NaN3 perfusion. After NaN3 perfusion, the Ach level in the model group stopped decreasing, but still be lower than that in the control all along. This result was similar to that of the previous one. During the whole observation, the Ach levels of 3 drug groups were higher than that of model. For RPMP and Duxil groups, comparaed with model, there were 2 time points showed significant differences respectively, while 7 points for RPMP & Duxil group. There was no significant meaning among the three drug groups on any time points.Compared to the control, the Ach area under content-time curve, minimum value of the model were significantly lower, but the situation was reversed for drug groups comparing to the model. However, the minimum value time was relative equal to each other between the model and drug groups.(2) The changes of Ch: While NaN3 perfusion starting, the extracellular Ch level in striatum of the model immediately increased singnificantly; and began to drop after NaN3 perfusion, then returned the same level of the control group 3h later. During the whole period, Ch levels of the drug groups were lower than that of the model. For RPMP group, comparaed with model, there were 3 time points showed significant differences, 6 for Duxil group and 1 for RPMP & Duxil group. There was no significant meaning among the three drug groups on any time pointsCompared to the control, the Ch area under content-time curve, maximum of the model were significantly higher, but the situation was reversed for drug groups comparing to the model. However, the maximum value time were relative equal to each other between the model and drug groups..(3) The activity of ChAT of the model group was significantly lower than that of the control while that of each drug group was significantly higher than model.(4) The activity of AchE was relatively equal among the all groups.(5) The activity of M-R of the model dropped compared to control, but the difference had no significance meaning. Compared to model, the activity of M-R
of Duxil and RPMP & Duxil groups increased significantly.(6) The activity of NMDA-R of the model increased compared to control, but the difference had no significance meaning. Compared to model, the activity of NMDA-R of each drug group decreased significantly.(7) The LD content in each group was relatively equal. 3.2.5 DiscussionThe results showed that the model was successfully repeated, since the extracellular Ach dropped obviously and the Ch level elevated obviously, which was relatively identical with the previous test.The three drug groups could improve the situation of Ach level decreasing caused by NaN3. This happened only during NaN3 perfusion in RPMP and Duxil groups, while was also generated when these two drugs used together in recovery period. Such improvement was aimed at decreasing degree, instead of delaying. Ch cumulating was reduced in each drug group, indicating the improvement of Ach level may relate to increasing the uptaking of extracellular Ch.The ChAT activity of the model decreased when 3.5h later after NaN3 perfusion, which may be caused by cell injure originated from the abnormity of oxidative phosphorylation in mitochondria caused by NaN3. Such change was coincidence with the change of Ach level, indicating Ach level decreasing may be caused by the dropping of ChAT activity. The drug groups could improve ChAT activity, showed their function of improving learning and memory were related to improving ChAT activity then causing Ach level increasing.The changes of AchE activity can not be a proof that NaN3 has no effects on AchE. This may be cause by neostigmine in the perfusion liqiud, which is an inhibitor of AchE.Duxil and RPMP & Duxil groups could increasing M-R activity, thus improve cholinergic nerve function, and approach the results of improving learning and memory function.The NMDA-R activity of model elevated when 3.5h after NaN3 perfusion compared to that of model group, but with on statistical significance. This
indicated that the influence of NaN3 was not enough to induce the over-releasing of EAA at this moment. But the anti-EAA toxic effects was showed in each drug group, indicating that they may decrease EAA toxic effects, protect cell and then improve Ach level.LD is a sensitive index for energy metabolism disorder, so it should appear quickly when cell damaged, also disappeared soon with the removal of damage factors. 3.5h after NaN3 perfusion, the level may have recovered to normal state, which led to the result of no obvious changes among groups. Hence, it is necessary to collect dialysate by microdialysis to dynamically detecting its changes so that to know the damage degree caused by NaN3, glucose and pyroracemic acid as well.3.2.6 Summary(1) Animal model was successfully repeated, since the extracellular Ach dropped obviously and the Ch level elevated obviously, which was relatively identical with the previous test. The ChAT activity of the model decreased, indicating the reason of Ach level decreasing may be caused by the dropping of ChAT activity.(2) The three drug groups could improve the situation of Ach level decreasing caused by NaN3.The effects were quite similar when RPMP or Duxil be used alone, while would be better when they were used together.(3) All 3 drug groups could decrease extracellular Ch level and improve intracellular ChAT activity, indicating they could improve the uptake of extracellular Ch so that evaluate the synthesizing material, and improve Ach synthesizing progress, resulting Ach increasing.(4) M-R activity was elevated and NMDA-R activity was reduced by the drug groups, indicating that they could keep cholinergic nerve function, abate cell damage caused by EAA over releasing, and approach their effects of brain protection.4. Conclusions(1) The extracellular Ach level in striatum decreased caused by NaN3 and
老年性痴呆(Alzheimer's Disease, AD)是严重危害老年人健康的疾病之一。脑内胆碱能神经退行性变导致AD认知功能缺损的观点,已被广泛接受。乙酰胆碱(Acetylcholine, Ach)是胆碱能神经传导的内源性化学递质,Ach脑内水平的平衡调节对维持学习、记忆、注意力等脑高级功能的正常运作至关重要。脑内微透析(Microdialysis)技术是一种在麻醉或清醒自由活动动物脑内取样的新手段,它的出现为脑内细胞外环境进行在体采样和动态监测提供了方便。
近年来,线粒体氧化磷酸化异常在AD发病中的作用引起人们的重视。叠氮钠(NaN_3)作为细胞色素C氧化酶抑制剂,能引起线粒体氧化磷酸化异常。研究表明,脑外给予NaN_3可导致动物学习记忆能力下降、胆碱能神经功能下降等与AD的病理改变相似的病变。本研究拟采用脑内灌流0.2%叠氮钠120min的方式造成胆碱能神经功能障碍的模型,为寻找新的治疗痴呆的药物提供可参考的方法。
何首乌是传统的常用的补益中药,有滋补肝肾,延年益寿,健脑调心的功效。已有的研究证实它具有改善学习记忆功能等药理作用,提示何首乌可能对脑有保护作用。本研究中选用已证实有效的剂量的制首乌水煎液进行研究。旨在探讨制首乌对叠氮钠灌流所致的大鼠脑内乙酰胆碱水平下降的调节作用及其机理。为何首乌作为脑保护剂治疗老年人常见的退行性疾病所致的痴呆提供实验依据。
2.研究方法
(1) 微透析技术动态监测脑内Ach/Ch水平变化:微透析技术是利用小分子物质能通过半透膜顺浓度梯度进行扩散的原理为基础进行脑内采样的技术。本研究采用此方法实现对清醒自由活动大鼠脑内细胞外Ach/Ch水
1. PurposeAlzheimer's Disease (AD) is one of the most severe diseases that damages the health of the senior citizen. It has been widely accepted that retrograde cholinergic nerve causes cognitive defect in AD patients. Acetylcholine (Ach) is the transmitter of cholinergic nerve, and the maintenance of balanced level of Ach in brain plays a important role in keeping the normal advanced function of brain, such as learning, memory and attention. Microdialysis is a new sampling method in brain of anaesthetized or conscious animals, and it facilitates sampling from brain extracellular environment in vivo and dynamiclly detecting.Recently, abnormal oxidative phosphorylation in mitochondria attracted much attention by its role in AD. Sodium azide (NaN_3), as an inhibitor of cytochrome oxidase (COX), can induce the abnormity of oxidative phosphorylation in mitochondria. Animal tests showed that NaN_3 administration outside of brain could reduce the learning and memory abilities, function of cholinergic function as well, which are similar to those of AD. In this study, we manage to made a animal model with reducing cholinergic function by NaN_3 perfusion 120 minutes in brain.Polygonum multiflorum Thumb. is a traditional material medica commonly used to nourish liver and kidney, prolong life, benefit brain and coordinate heart. It has been proved that it has the function of improving learning and memory abilities, which indicated that it may protect brain. In this study, the confirmed
effective dose of decoct of Radix Polygoni Multiflori Preparata (RPMP) was used to find out its function and mechanism on adjusting the Ach level in rat's brain with NaN3 perfusion. It may provide the experimental evidence to support the idea that Polygonum multiflorum Thumb, could be brain protective drug to treat retrogressive disease such as dementia.2. Method2.1 Dynamically inspecting on the changes of Ach/Ch levels in brain with microdialysis: Microdialysis is a kind of sampling technique in brain which based on the principle that small molecule can penetrate semipermeable membranes. In this study, the method was used for dynamically detecting on the changes of Ach/Ch levels in the brain of conscious free moving rats.2.2 Determining the content of Ach and Ch in dialysate with the method of high performance liquid chromatography - Immobilized enzyme reactor -electrochemical detection (HPLC-IMER-ED): Based on the operation manual( MF-9053 ) from BAS company and related literatures, this method was used to determine the content of Ach/Ch which was collected by Microdialysis collection. Methodology test show that it is sensitive and reliable.2.3 Determining the activity of choline-acetyl-transfertase (ChAT) with radio isotope biding method.2.4 Determining the activity of Acetylcholinesterase (AchE) with spectrophotometry2.5 Determining the binding ability of cholinergic M receptor (M-R) and N-methyl-D-aspartic acid receptor (NMDA-R) with radioactive aglucone -receptor biding method2.6 Determining the content of LD with spectrophotometry3. Content3.1 Study on the animal model with reducing cholinergic function by NaN3 perfusion 120 minutes in brain.
3.1.1 Groups and treatments of animalsSD rats were randomly divided into two groups: the control group and the model group.Protocol of Microdialysis: implanted a probe into striatum for perfusion and collected one vial of dialysate per 30 min. The process could be divided into 3 steps for the model rats: pre-perfusion of NaN3, perfusion of NaN3 (2 h) and after NaN3 perfusion (16.5 h, recovery period), while the control group rats were perfused with Ringer solution all along.3.1.2 Index of research? The content of Ach and Ch indialysate. ? decollate rats after microdialysis progress and remove the striatum, then determined the activity of ChAT, AchE, M-R and NMDA-R, the content of lactic acid (LD) as well.3.1.3 Statistical analysisThe data of the two groups were showed with Mean ± Sd, and t-test was used to compare mean value3.1.4 Results(1) The changes of Ach: During the whole observation period, the extracellular Ach level of the control group in striatum stayed stable, with fluctuation occasionally. While the level of the model rats dropped greatly during the period of NaN3 perfusion. The Ach level stopped dropping after NaN3 perfusion, it stayed stable and showed the tendency of increasing slowly from 5.5h later, and at 14.5h in recovery period, the Ach level reached relatively equal to that of control group.(2) The changes of Ch level: The Ch level of the control group stayed stable during the whole period of microdialysis. However, the Ch level of model increased rapidly immediately when NaN3 perfusion starting, reached its peak lh later, then entered the platform phrase after this. After NaN3 perfusion the Ch level began to drop, and stayed stable 4h later.(3) The changes of other indexes: 16.5 h later after NaN3 perfusion, the activity of ChAT and M-R in the model group decreased with no statistical
significance, compared to those of the control. AchE activity and the content of lactic acid of the two groups were very close to each other. The activity of NMDA-R in the model group increased significantly.3.1.5 DiscussionThe extracellular Ach level in the brain can mirror the cholinergic function in brain reliably. The results showed the extracellular Ach level in striatum decreased caused by 0.2% NaN3 perfusion 120min through microdialysis administration, and this changes were reversible with its level return to the normal value 14.5h after NaN3 perfusion. So, the animal model was successfully made.Ch is the material of synthesizing Ach, and its cleavage product as well. This study showed extracellular Ch level increased significantly caused by NaN3. Such changes may relate to the uptake disorder of Ch, which result the decreasing synthesis of Ach in neurons.The study on neurotransmitter can not be limited to the changes itself, but the analysis of its receptor and related enzymes should also be conducted to make clear the changes of transmitter system on a wide scale. The activity of ChAT, AchE and M-R did not showed the significantly changes in this test. The content of LD, having the function of reflecting mitochondria function decreasing, did not change significantly either. Such negative results may relate to the time of sampling: the sampling began at 16.5h after NaN3 perfusion, when these changes had weaken even disappeared.The toxic effects of excitatory amino acid (EAA) induced by super-activation of NMDA-R plays an important role in retrograde neuropathogenesis such as AD. This study showed the activity of NMDA-R increased significantly caused by NaN3 perfusion.3.1.6 Summary(1) In this study, the dynamically changes of extracellular Ach and Ch in striatum during 0.2% NaN3 perfusion 120 min and recovery period were observed in a long period. The results of reversible decreasing of Ach level and increasing Ch level indicated the animal model that imitate the reducing of
cholinergic nerve function was successfully made.(2) The negative results indicated that the damage may have recovered when sampling conducted at 16.5h after NaN3 perfusion.. Based on the results of Ach and Ch, we plan to decollate animals and remove striatum when 3.5 h after NaN3 perfusion in order to observe the changes of relative indexes in the later research..3.2 Study on Protection Mechanism of Radix Polygoni Multiflori Preparata in Rats Brain with Sodium Azide Perfusion3.2.1 Groups and treatmentsSD rats were randomly divided into 5 groups: RPMP group (15g/Kg), Duxil group (lOmg/Kg), RPMP & Duxil group (RPMP 15g/Kg & Duxil lOmg/Kg), Control and Model (distilled water). Followed by the above doses, rats were i.g. for 14 days continuously. On the 13th day, guide tube was implanted into the brain of rat and then the rats were operated with microdialysis the next day.Microdialysis protocol: implanted a probe into striatum for perfusion and collected one vial of dialysate per 30 min. Control was perfused with Ringer solution all along. Process could be divided into 3 steps for the other groups: pre-perfusion of NaN3, perfusion of NaN3 (2 h) and after NaN3 perfusion (3.5 h, recovery period).3.2.2 Index of research? The content of Ach and Ch in dialysate, their area under the content-time curve, extreme values and the extreme time. @ decollate rats after Microdialysis progress and remove the striatum, then determined the activity of ChAT, AchE, M-R and NMDA-R, the content of lactic acid (LD) as well.3.2.3 Statistical analysisIndex of each group was showed with Mean ± Sd. ANOVA was used, and if there is significant difference then use LSD method to compare the average among goups.3.2.4 Results
(1) Changes of Ach: The extracellular Ach level of striatum stayed stable in the control group during the whole observing period but dropped significantly in the model group during NaN3 perfusion. After NaN3 perfusion, the Ach level in the model group stopped decreasing, but still be lower than that in the control all along. This result was similar to that of the previous one. During the whole observation, the Ach levels of 3 drug groups were higher than that of model. For RPMP and Duxil groups, comparaed with model, there were 2 time points showed significant differences respectively, while 7 points for RPMP & Duxil group. There was no significant meaning among the three drug groups on any time points.Compared to the control, the Ach area under content-time curve, minimum value of the model were significantly lower, but the situation was reversed for drug groups comparing to the model. However, the minimum value time was relative equal to each other between the model and drug groups.(2) The changes of Ch: While NaN3 perfusion starting, the extracellular Ch level in striatum of the model immediately increased singnificantly; and began to drop after NaN3 perfusion, then returned the same level of the control group 3h later. During the whole period, Ch levels of the drug groups were lower than that of the model. For RPMP group, comparaed with model, there were 3 time points showed significant differences, 6 for Duxil group and 1 for RPMP & Duxil group. There was no significant meaning among the three drug groups on any time pointsCompared to the control, the Ch area under content-time curve, maximum of the model were significantly higher, but the situation was reversed for drug groups comparing to the model. However, the maximum value time were relative equal to each other between the model and drug groups..(3) The activity of ChAT of the model group was significantly lower than that of the control while that of each drug group was significantly higher than model.(4) The activity of AchE was relatively equal among the all groups.(5) The activity of M-R of the model dropped compared to control, but the difference had no significance meaning. Compared to model, the activity of M-R
of Duxil and RPMP & Duxil groups increased significantly.(6) The activity of NMDA-R of the model increased compared to control, but the difference had no significance meaning. Compared to model, the activity of NMDA-R of each drug group decreased significantly.(7) The LD content in each group was relatively equal. 3.2.5 DiscussionThe results showed that the model was successfully repeated, since the extracellular Ach dropped obviously and the Ch level elevated obviously, which was relatively identical with the previous test.The three drug groups could improve the situation of Ach level decreasing caused by NaN3. This happened only during NaN3 perfusion in RPMP and Duxil groups, while was also generated when these two drugs used together in recovery period. Such improvement was aimed at decreasing degree, instead of delaying. Ch cumulating was reduced in each drug group, indicating the improvement of Ach level may relate to increasing the uptaking of extracellular Ch.The ChAT activity of the model decreased when 3.5h later after NaN3 perfusion, which may be caused by cell injure originated from the abnormity of oxidative phosphorylation in mitochondria caused by NaN3. Such change was coincidence with the change of Ach level, indicating Ach level decreasing may be caused by the dropping of ChAT activity. The drug groups could improve ChAT activity, showed their function of improving learning and memory were related to improving ChAT activity then causing Ach level increasing.The changes of AchE activity can not be a proof that NaN3 has no effects on AchE. This may be cause by neostigmine in the perfusion liqiud, which is an inhibitor of AchE.Duxil and RPMP & Duxil groups could increasing M-R activity, thus improve cholinergic nerve function, and approach the results of improving learning and memory function.The NMDA-R activity of model elevated when 3.5h after NaN3 perfusion compared to that of model group, but with on statistical significance. This
indicated that the influence of NaN3 was not enough to induce the over-releasing of EAA at this moment. But the anti-EAA toxic effects was showed in each drug group, indicating that they may decrease EAA toxic effects, protect cell and then improve Ach level.LD is a sensitive index for energy metabolism disorder, so it should appear quickly when cell damaged, also disappeared soon with the removal of damage factors. 3.5h after NaN3 perfusion, the level may have recovered to normal state, which led to the result of no obvious changes among groups. Hence, it is necessary to collect dialysate by microdialysis to dynamically detecting its changes so that to know the damage degree caused by NaN3, glucose and pyroracemic acid as well.3.2.6 Summary(1) Animal model was successfully repeated, since the extracellular Ach dropped obviously and the Ch level elevated obviously, which was relatively identical with the previous test. The ChAT activity of the model decreased, indicating the reason of Ach level decreasing may be caused by the dropping of ChAT activity.(2) The three drug groups could improve the situation of Ach level decreasing caused by NaN3.The effects were quite similar when RPMP or Duxil be used alone, while would be better when they were used together.(3) All 3 drug groups could decrease extracellular Ch level and improve intracellular ChAT activity, indicating they could improve the uptake of extracellular Ch so that evaluate the synthesizing material, and improve Ach synthesizing progress, resulting Ach increasing.(4) M-R activity was elevated and NMDA-R activity was reduced by the drug groups, indicating that they could keep cholinergic nerve function, abate cell damage caused by EAA over releasing, and approach their effects of brain protection.4. Conclusions(1) The extracellular Ach level in striatum decreased caused by NaN3 and
引文
1.赖荣汉,刘晓文,王毅兵.何首乌炮制沿革与现代研究.中国中医药信息杂志,1999,6(9):19
2.周燕华.“白”何首乌的考证.中国中药杂志,1999,24(4):243-145
3.王惠群,詹国瑛.何首乌的诱变性研究.贵阳医学院学报,2002,27(1):22-23
4.楚晋,叶翠飞,李林.二苯乙烯苷对痴呆小鼠学习记忆及自由基代谢的影响.中国康复理论与实践,2003,9(11):643-645
5.叶翠飞,张兰,李斌,等.二苯乙烯苷对胆碱能损伤拟老年性痴呆大鼠学习记忆功能和兴奋性的影响.中国康复理论与实践,2003,9(10):593-595
6.刘治军,李林,叶翠飞,等.二苯乙烯苷对脑缺血小鼠脑组织含水量及自由基代谢的影响.中国康复理论与实践,2003,9(11):641-642
7.刘治军,李林,叶翠飞,等.二苯乙烯苷对脑缺血啮齿动物脑N M D A受体及细胞内钙离子的影响.中国药理学通报,2003,19(10):1112～1115
8.张兰,李林,李雅莉.何首乌有效成分二苯乙烯甙对神经细胞保护作用的机制.中国临床康复,2004,8(1):118-120
9.陈万生,徐江平,李力,等.大黄素-8-O-β-D-吡哺葡萄糖苷的促智活性及其机制.中草药,2001,32(1):39-41
10.谭佩珍,杨期东,谭兴林,等.何首乌治疗血管性痴呆的疗效观察.河南医药信息,2002,10(12):7-9
11.徐承水.何首乌提取物对大鼠过氧化损伤的保护作用.烟台师范学院学报(自然科学版),2000,16(3):197-199
12.侯德仁,杨期东,周琳,等.何首乌对Alzheimer病模型大鼠学习记忆的影响及其机制的研究.中国医师杂志,2004,6(3):347-349
13.李文,杜小平,叶晖,等.何首乌对KA致大鼠脑胆碱能纤维损伤的保护作用.湖南医科大学学报,2003,28(4):361-364
14.莫启忠,宫斌,方军,等.补肾中药对SRBC免疫后老年大鼠脑组织中M和β受体功能的影响.中国免疫学杂志,1997,13(2):90-93
15.袁崇刚,白桦,阳飞昆.首乌制剂对MPTP引起的小鼠帕金森病的防治.华东师范大学学报(自然科学版),2002,(3):95-98
16.姚谦明,蒋宇刚,何启.何首乌对脑细胞Bcl-2基因表达的影响实验性研究.现代临床医学生物工程学杂志,2002,8(2):83-86
17.张志英,刘荣建,王绍坤.下丘脑室周核内生长抑素能神经元的衰老变化及何首乌的抗衰老作用.中华老年医学杂志,1996,15(4):223-225
18.张兰,李林,李斌,等.中药参乌胶囊对老年大鼠学习记忆及海马组织学的影响.中国康复理论与实践,2002,8(9):516-519
19.王辉,李林,叶翠飞,等.参乌胶囊对淀粉样蛋白大鼠模型外周血淋巴细胞DNA的影响.中国中药杂志,2004,29(2):176-178
20.胡镜清,赖世隆,王奇,等.补肾益智方对阿尔茨海默病大鼠模型脑内胆碱能神经系统的保护作用.广州中医药大学学报,1999,16(3):201-204
21.胡镜清,赖世隆,王奇,等.补肾益智方阻抑老年性痴呆大鼠海马突触病理性重构的研究.广州中医药大学学报,2000,17(2):113-116
22.马宜明,杜贺庆.首乌神颗粒对小鼠学习记忆能力的影响.中药药理与临床,2001,17(4):35-37
23.马宜明,李俊,金涌.首乌神颗粒改善学习记忆的部分机制研究.安徽医药,2003,7(1):7-9
24.唐一鹏,孙承琳,洪庆涛,等.中药“健脑液”对培养神经细胞迟发性损伤的保护作用.北京中医药大学学报,1995,18(2):26-29
25.苗明三,方晓艳.制何首乌多糖对衰老模型小鼠抗氧化作用的研究.中药药理与临床,2002,18(5):23-24
26.韩志芬,顾文聪.不同首乌提取液对老年大鼠心、肝、脑自由基代谢及脑内MAO-B的影响.中国中医药科技,1995,2(1):36-37
27.周晔,李一峻,陈强,等.覆盆子等8味中药的抗超氧阴离子自由基作用研究.时珍国医国药,2004,15(2):68-69
28.李丽帆,刘官和.首乌提取液对O_2~-的清除作用.右江民族医学院学报,1997(3):360-361
29.吴晓光,杨松鹤,陈志宏.何首乌颗粒抗自由基作用的实验研究.承德医学院学报, 2004,21(1):3-5
30.钱汝红,丁镛发,严军,等.首乌对老年小鼠脾组织中抗癌基因P53表达的影响.中国中医药科技,1999,6(2):89-90
31.金国琴,赵伟康.首乌制剂对老年大鼠胸腺、肝脏蛋白质和核酸含量的影响.中草药,1994,25(11):590-591
32.魏锡云,张锦,罗映辉,等.黄芪和何首乌抗环磷酰胺诱导胸腺细胞凋亡.中国药科大学学报,2000,31(1):35-38
33.赵伟康,金国琴.固真方对老年大鼠胸腺细胞糖皮质激素受体作用的研究.中国中西医结合杂志,1995,15(2):92-94
34.赵伟康,凌昌全,金国琴,等.固真方对~(60)Co-γ射线照射大鼠神经内分泌及免疫系统的调节作用.中华放射医学与防护杂志,1994,14(6):376-378
35.黄青松.脑力康对老年大鼠血清白细胞介素-2水平的影响.中国中医药科技,1999,6(3):143
36.张黎,芮耀诚,邱彦,等.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯2-O-β-D-葡糖苷(STI)对内皮细胞表VEGF的影响.药学学报,2004,39(6):406-409
37.刘厚淳,陈万生.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯2-O-β—D-葡糖苷的体外抗氧化作用研究.药学实践杂志,2000,1 8(4):232-233
38.陈万生,刘文庸,杨根金,等.制首乌中1个新的四羟基二苯乙烯苷的结构鉴定及其心血管活性研究.药学学报,2000,35(12):906-908
39.刘彦珠,罗国安,龙致贤,等.首乌及其复方对平滑肌细胞增殖抑制作用的研究.清华大学学报(自然科学版),1998,38(12):32-35
40.徐品初,林水淼,陈方敏,等.补肾益气方及其拆方对氧化低密度脂蛋白损伤血管内皮细胞的影响.现代中两医结合杂志,2004,13(16):2129-2130
41.王宝珠,陈锡林.“首乌降脂汤”对大鼠食物性高脂血症的预防作用研究.中国野生植物资源,1999,18(2):40-42
42.王桂敏,吴秀青.首乌延寿丹抗血管内皮细胞老化的实验研究.中医药学刊,2002,20(3):314-315
43.张九方,程学萱,仲肇书,等.动脉粥样硬化防治新途径的初步探索.南京医科大学学报,1995,15(2):333-335
44.孟扬,王瑞绵,严四新.复方首乌对受损内皮促修复及释放PGI2的作用.湖北医科大学学报,1998,19(2):97-100
45.王钢,仲昱,孔薇,等.保肾片对食盐敏感性高血压大鼠肾损害的保护.中华肾脏病杂志,2003,19(3):185-186
46.张笑丽,韩景辉,杨瑞霞,等.首乌降压胶囊对肾性高血压大鼠肾素-血管紧张素-醛固酮系统作用的实验研究.辽宁中医杂志,2004,31(8):705-706
47.张笑丽,杨瑞霞,刘子旺,等.首鸟降压胶囊对肾性高血压大鼠血浆ANP、CGRP的影响.中国医药学报,2004,19(11):694-695
48.陈可冀主编.跨世纪脑科学:老年性痴呆发病机理与诊治.第一版.北京:北京医科大学中国协和医科大学联合出版社,1998:263-282
49.阎孝诚主编.实用中医脑病学.第1版.天津:天津科学技术出版社.1994:55-66
50. Benveniste H, Drejer J, Schousboe A, et al. Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. J Neurochem. 1987,49(3):729-734
51. Westerink BH, De Vries JB. Characterization of in vivo dopamine release as determined by brain microdialysis after acute and subchronic implantations: methodological aspects. J Neurochem. 1988,51(3):683-687
52. Benveniste H, Diemer NH. Cellular reactions to implantation ofa microdialysis tube in the rat hippocampus. Acta Neuropathol (Berl). 1987;74(3):234-238
53. Shuaib A, Xu K, Crain B, et al. Assessment of damage from implantation of microdialysis probes in the rat hippocampus with silver degeneration staining. Neurosci Lett. 1990,112(2-3):149-154
54. Hamberger A, Nystrom B. Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy. Neurochem Res. 1984,9(9):1181-1192
55. Landers JP. Clinical capillary electrophoresis. Clin Chem. 1995,41(4):495-509
56. Callaghan DH, Yergey JA, Rousseau P, et al. Respiratory tract eicosanoid measurement using microdialysis sampling and GC/MS detection. Pulm Pharmacol. 1994,7(1):35-41
57.蔡晓辉,王广基.生物传感技术在体内药代动力学研究中的应用.药学进展,2001,25(3)142-145
58.汪海林,邹汉法,张玉奎.微透析—液相色谱法研究药物与蛋白竞争结合.中国科学(B辑),1998,28(1):71-77
59. Wages SA, Church WH, Justice JB Jr. Sampling considerations for on-line microbore liquid chromatography of brain dialysate. Anal Chem. 1986,58(8):1649-1656
60. Chaurasia CS. In vivo microdialysis sampling: theory and applications. Biomed Chromatogr. 1999,13(5):317-332
61. Johnson RD, Justice JB. Model studies for brain dialysis. Brain Res Bull, 1983,10(4):567-571
62. Benveniste H, Hansen AJ, Ottosen NS. Determination of brain interstitial concentrations by microdialysis. J Neurochem. 1989,52(6):1741-1750
63. Stahle L, Segersvard S, Ungerstedt U. A comparison between three methods for estimation of extracellular concentrations of exogenous and endogenous compounds by microdialysis. J Pharmacol Methods. 1991,25(1):41-52
64. Menacherry S, Hubert W, Justice JB Jr. In vivo calibration ofmicrodialysis probes for exogenous compounds. Anal Chem. 1992,64(6):577-583.
65. Hsiao JK, Ball BA, Mon-ison PF, et al. Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes. J Neurochem. 1990,54(4): 1449-1452
66. Tsai TH, Liang C. Pharmacokinetics of tetramethylpyrazine in rat blood and brain using microdialysis. Int J Pharm. 2001,216(1-2):61-66
67. Westerink BH, Hofsteede HM, Damsma G, et al. The significance of extracellular calcium for the release of dopamine, acetylcholine and amino acids in conscious rats, evaluated by brain microdialysis. Naunyn Schmiedebergs Arch Pharmacol. 1988,337(4):373-378
68. Osborne PG, O'Connor WT, Ungerstedt U. Effect of varying the ionic concentration of a microdialysis perfusate on basal striatal dopamine levels in awake rats. J Neurochem. 1991,56(2):452-456
69.赵秋华,岳云,于代华,等.氯胺酮和咪唑安定麻醉下脑皮层谷氨酸含量的变化:活体脑微透析研究.临床麻醉学杂志,2001,17(7):381-384
70.伍建林.氯胺酮对鼠纹状体乙酰胆碱释放的影响.中山医科大学学报,1996,17(3):228-230
71.贾兴元,吴安石,岳云,等.微柱高效液相色谱法测定大鼠脑微透析液中的乙酰胆碱和胆碱.色谱,2004,22(1):33-35
72.陈生弟主编,神经变性性疾病.第1版,人民军医出版社,北京.2002:265
73.张兰,李林.线粒体缺陷与阿尔采末病.生理科学进展.1999;30(4):363-366
74.张兰,李林,刘树森,等.叠氮钠对培养神经细胞微管结构和细胞存活率的影响.中国神经科学杂志,2000,16(3):257-262
75.张兰,李林,班立勤,等.叠氮钠对SH-SY5Y人神经母细胞瘤细胞线粒体跨膜电位的影响.中国医学科学院学报.2000;22(5):436-439
76.张兰,叶翠飞,张如意,等.微泵恒速灌注叠氮钠对大鼠学习记忆的影响.中国行为医学科学.2001,10(1):1-3
77.张林,等.微泵恒速灌注叠氮钠对大鼠学习记忆及胆碱乙酰转移酶活性的影响.中国药理学会通讯.2000,17(2):44-45
78.王本祥主编.现代中药药理学.第一版.天津科学技术出版社.天津.1999:1306
79.王巍,等.何首乌及其提取物对小鼠记忆获得障碍的影响.中国中两医结合杂志.1999;19(特):26-28
80.李旻,杜小平,叶晖,等.何首乌对CK所致大鼠脑胆碱能纤维损伤的保护作用.脑与神经疾病杂志.2002,10(3):137-139
81.王巍,赵德忠,孙晓芳,等.中药首茸方对左旋多巴诱发帕金森病大鼠脑内羟自由基增高的影响.中国中西医结合杂志,2003,23(增刊):50-53
82. Lowry OH, et al. Protein mesurement with folin-phenyl agent. J Biol Chem. 1951,193(1):265
83. Huang T, Yang L, Gitzen J et al. Detection of basal acetylcholine in rat brain microdialysate. J Chromatogr B Biomed Appl, 1995,670(2): 323-327.
84. Greaney MD, Marshall DL, Bailey BA, et al. Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor. J Chromatogr, 1993, 622(2): 125-135.
85. Parker WD Jr, Filly CM, Parks JK. Cytochrome oxidase deficiency in Alzheimer's disease. Neurology, 1990, 40(8): 1302-1303.
86. Parker WD, Mahr NJ, Filly CM, et al. Reduced Platelet Cytochrome C oxidase activity in Alzheimer's disease. Neurology, 1994, 44(6): 1086-1090
87. Parker WD Jr, Parks JK. Cytochrome c oxidase in Alzheimer's disease brain: purification and characterization. Neurology, 1995, 45(3 Pt 1): 482-486
88. Mutisya EM, Bowling AC, Beal ME Cortical cytohrome oxidase activity is reduced in Alzhermer's disease. J Neurochem, 1994, 63(6): 2179-2184.
89. Bennett MC, Diamond DM, Stryker SL, et al. Cytochrome oxidase inhibition: a novel animal model of Alzheimer's disease. J Geriatr Psychiatry Neurol. 1992;5(2):93-101
90. Bennett MC, Rose GM. Chronic sodium azide treatment impairs learning of the Morris water maze task. Behav Neural Biol. 1992,58(1):72-75
91.徐海伟,黎海蒂,范晓棠,等.AD大鼠模型的制备及行为学和超微结构研究.中国行为医学科学,2003,12(2):123-127
92. Smith TS, Bennett JP Jr. Mitochondrial toxins in models of neurodegenerative diseases. Ⅰ: In vivo brain hydroxyl radical production during systemic MPTP treatment or following microdialysis infusion of methyllpyridinium or azide ions. Brain Res, 1997, 765(2): 183-188
93.徐海伟,黎海蒂,范晓棠,等.褪黑素在叠氮钠所致的海马锥体细胞钙超载中的神经保护作用.第三军医大学报,2003,25(3):201-204.
94.张兰,张如意,李林,等馓泵恒速灌注叠氮钠致痴呆大鼠脑内胆碱及单胺类神经递质含量改变.中国康复理论与实践,2003,9(9):526-529
95. Nicoletti F, Canonico PL. Glycine potentiates the stimulation of inositol phospholipid hydrolysis by excitatory amino acids in primary cultures of cerebellar neurons. J Neurochem. 1989,53(3):724-727.
2.周燕华.“白”何首乌的考证.中国中药杂志,1999,24(4):243-145
3.王惠群,詹国瑛.何首乌的诱变性研究.贵阳医学院学报,2002,27(1):22-23
4.楚晋,叶翠飞,李林.二苯乙烯苷对痴呆小鼠学习记忆及自由基代谢的影响.中国康复理论与实践,2003,9(11):643-645
5.叶翠飞,张兰,李斌,等.二苯乙烯苷对胆碱能损伤拟老年性痴呆大鼠学习记忆功能和兴奋性的影响.中国康复理论与实践,2003,9(10):593-595
6.刘治军,李林,叶翠飞,等.二苯乙烯苷对脑缺血小鼠脑组织含水量及自由基代谢的影响.中国康复理论与实践,2003,9(11):641-642
7.刘治军,李林,叶翠飞,等.二苯乙烯苷对脑缺血啮齿动物脑N M D A受体及细胞内钙离子的影响.中国药理学通报,2003,19(10):1112～1115
8.张兰,李林,李雅莉.何首乌有效成分二苯乙烯甙对神经细胞保护作用的机制.中国临床康复,2004,8(1):118-120
9.陈万生,徐江平,李力,等.大黄素-8-O-β-D-吡哺葡萄糖苷的促智活性及其机制.中草药,2001,32(1):39-41
10.谭佩珍,杨期东,谭兴林,等.何首乌治疗血管性痴呆的疗效观察.河南医药信息,2002,10(12):7-9
11.徐承水.何首乌提取物对大鼠过氧化损伤的保护作用.烟台师范学院学报(自然科学版),2000,16(3):197-199
12.侯德仁,杨期东,周琳,等.何首乌对Alzheimer病模型大鼠学习记忆的影响及其机制的研究.中国医师杂志,2004,6(3):347-349
13.李文,杜小平,叶晖,等.何首乌对KA致大鼠脑胆碱能纤维损伤的保护作用.湖南医科大学学报,2003,28(4):361-364
14.莫启忠,宫斌,方军,等.补肾中药对SRBC免疫后老年大鼠脑组织中M和β受体功能的影响.中国免疫学杂志,1997,13(2):90-93
15.袁崇刚,白桦,阳飞昆.首乌制剂对MPTP引起的小鼠帕金森病的防治.华东师范大学学报(自然科学版),2002,(3):95-98
16.姚谦明,蒋宇刚,何启.何首乌对脑细胞Bcl-2基因表达的影响实验性研究.现代临床医学生物工程学杂志,2002,8(2):83-86
17.张志英,刘荣建,王绍坤.下丘脑室周核内生长抑素能神经元的衰老变化及何首乌的抗衰老作用.中华老年医学杂志,1996,15(4):223-225
18.张兰,李林,李斌,等.中药参乌胶囊对老年大鼠学习记忆及海马组织学的影响.中国康复理论与实践,2002,8(9):516-519
19.王辉,李林,叶翠飞,等.参乌胶囊对淀粉样蛋白大鼠模型外周血淋巴细胞DNA的影响.中国中药杂志,2004,29(2):176-178
20.胡镜清,赖世隆,王奇,等.补肾益智方对阿尔茨海默病大鼠模型脑内胆碱能神经系统的保护作用.广州中医药大学学报,1999,16(3):201-204
21.胡镜清,赖世隆,王奇,等.补肾益智方阻抑老年性痴呆大鼠海马突触病理性重构的研究.广州中医药大学学报,2000,17(2):113-116
22.马宜明,杜贺庆.首乌神颗粒对小鼠学习记忆能力的影响.中药药理与临床,2001,17(4):35-37
23.马宜明,李俊,金涌.首乌神颗粒改善学习记忆的部分机制研究.安徽医药,2003,7(1):7-9
24.唐一鹏,孙承琳,洪庆涛,等.中药“健脑液”对培养神经细胞迟发性损伤的保护作用.北京中医药大学学报,1995,18(2):26-29
25.苗明三,方晓艳.制何首乌多糖对衰老模型小鼠抗氧化作用的研究.中药药理与临床,2002,18(5):23-24
26.韩志芬,顾文聪.不同首乌提取液对老年大鼠心、肝、脑自由基代谢及脑内MAO-B的影响.中国中医药科技,1995,2(1):36-37
27.周晔,李一峻,陈强,等.覆盆子等8味中药的抗超氧阴离子自由基作用研究.时珍国医国药,2004,15(2):68-69
28.李丽帆,刘官和.首乌提取液对O_2~-的清除作用.右江民族医学院学报,1997(3):360-361
29.吴晓光,杨松鹤,陈志宏.何首乌颗粒抗自由基作用的实验研究.承德医学院学报, 2004,21(1):3-5
30.钱汝红,丁镛发,严军,等.首乌对老年小鼠脾组织中抗癌基因P53表达的影响.中国中医药科技,1999,6(2):89-90
31.金国琴,赵伟康.首乌制剂对老年大鼠胸腺、肝脏蛋白质和核酸含量的影响.中草药,1994,25(11):590-591
32.魏锡云,张锦,罗映辉,等.黄芪和何首乌抗环磷酰胺诱导胸腺细胞凋亡.中国药科大学学报,2000,31(1):35-38
33.赵伟康,金国琴.固真方对老年大鼠胸腺细胞糖皮质激素受体作用的研究.中国中西医结合杂志,1995,15(2):92-94
34.赵伟康,凌昌全,金国琴,等.固真方对~(60)Co-γ射线照射大鼠神经内分泌及免疫系统的调节作用.中华放射医学与防护杂志,1994,14(6):376-378
35.黄青松.脑力康对老年大鼠血清白细胞介素-2水平的影响.中国中医药科技,1999,6(3):143
36.张黎,芮耀诚,邱彦,等.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯2-O-β-D-葡糖苷(STI)对内皮细胞表VEGF的影响.药学学报,2004,39(6):406-409
37.刘厚淳,陈万生.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯2-O-β—D-葡糖苷的体外抗氧化作用研究.药学实践杂志,2000,1 8(4):232-233
38.陈万生,刘文庸,杨根金,等.制首乌中1个新的四羟基二苯乙烯苷的结构鉴定及其心血管活性研究.药学学报,2000,35(12):906-908
39.刘彦珠,罗国安,龙致贤,等.首乌及其复方对平滑肌细胞增殖抑制作用的研究.清华大学学报(自然科学版),1998,38(12):32-35
40.徐品初,林水淼,陈方敏,等.补肾益气方及其拆方对氧化低密度脂蛋白损伤血管内皮细胞的影响.现代中两医结合杂志,2004,13(16):2129-2130
41.王宝珠,陈锡林.“首乌降脂汤”对大鼠食物性高脂血症的预防作用研究.中国野生植物资源,1999,18(2):40-42
42.王桂敏,吴秀青.首乌延寿丹抗血管内皮细胞老化的实验研究.中医药学刊,2002,20(3):314-315
43.张九方,程学萱,仲肇书,等.动脉粥样硬化防治新途径的初步探索.南京医科大学学报,1995,15(2):333-335
44.孟扬,王瑞绵,严四新.复方首乌对受损内皮促修复及释放PGI2的作用.湖北医科大学学报,1998,19(2):97-100
45.王钢,仲昱,孔薇,等.保肾片对食盐敏感性高血压大鼠肾损害的保护.中华肾脏病杂志,2003,19(3):185-186
46.张笑丽,韩景辉,杨瑞霞,等.首乌降压胶囊对肾性高血压大鼠肾素-血管紧张素-醛固酮系统作用的实验研究.辽宁中医杂志,2004,31(8):705-706
47.张笑丽,杨瑞霞,刘子旺,等.首鸟降压胶囊对肾性高血压大鼠血浆ANP、CGRP的影响.中国医药学报,2004,19(11):694-695
48.陈可冀主编.跨世纪脑科学:老年性痴呆发病机理与诊治.第一版.北京:北京医科大学中国协和医科大学联合出版社,1998:263-282
49.阎孝诚主编.实用中医脑病学.第1版.天津:天津科学技术出版社.1994:55-66
50. Benveniste H, Drejer J, Schousboe A, et al. Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. J Neurochem. 1987,49(3):729-734
51. Westerink BH, De Vries JB. Characterization of in vivo dopamine release as determined by brain microdialysis after acute and subchronic implantations: methodological aspects. J Neurochem. 1988,51(3):683-687
52. Benveniste H, Diemer NH. Cellular reactions to implantation ofa microdialysis tube in the rat hippocampus. Acta Neuropathol (Berl). 1987;74(3):234-238
53. Shuaib A, Xu K, Crain B, et al. Assessment of damage from implantation of microdialysis probes in the rat hippocampus with silver degeneration staining. Neurosci Lett. 1990,112(2-3):149-154
54. Hamberger A, Nystrom B. Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy. Neurochem Res. 1984,9(9):1181-1192
55. Landers JP. Clinical capillary electrophoresis. Clin Chem. 1995,41(4):495-509
56. Callaghan DH, Yergey JA, Rousseau P, et al. Respiratory tract eicosanoid measurement using microdialysis sampling and GC/MS detection. Pulm Pharmacol. 1994,7(1):35-41
57.蔡晓辉,王广基.生物传感技术在体内药代动力学研究中的应用.药学进展,2001,25(3)142-145
58.汪海林,邹汉法,张玉奎.微透析—液相色谱法研究药物与蛋白竞争结合.中国科学(B辑),1998,28(1):71-77
59. Wages SA, Church WH, Justice JB Jr. Sampling considerations for on-line microbore liquid chromatography of brain dialysate. Anal Chem. 1986,58(8):1649-1656
60. Chaurasia CS. In vivo microdialysis sampling: theory and applications. Biomed Chromatogr. 1999,13(5):317-332
61. Johnson RD, Justice JB. Model studies for brain dialysis. Brain Res Bull, 1983,10(4):567-571
62. Benveniste H, Hansen AJ, Ottosen NS. Determination of brain interstitial concentrations by microdialysis. J Neurochem. 1989,52(6):1741-1750
63. Stahle L, Segersvard S, Ungerstedt U. A comparison between three methods for estimation of extracellular concentrations of exogenous and endogenous compounds by microdialysis. J Pharmacol Methods. 1991,25(1):41-52
64. Menacherry S, Hubert W, Justice JB Jr. In vivo calibration ofmicrodialysis probes for exogenous compounds. Anal Chem. 1992,64(6):577-583.
65. Hsiao JK, Ball BA, Mon-ison PF, et al. Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes. J Neurochem. 1990,54(4): 1449-1452
66. Tsai TH, Liang C. Pharmacokinetics of tetramethylpyrazine in rat blood and brain using microdialysis. Int J Pharm. 2001,216(1-2):61-66
67. Westerink BH, Hofsteede HM, Damsma G, et al. The significance of extracellular calcium for the release of dopamine, acetylcholine and amino acids in conscious rats, evaluated by brain microdialysis. Naunyn Schmiedebergs Arch Pharmacol. 1988,337(4):373-378
68. Osborne PG, O'Connor WT, Ungerstedt U. Effect of varying the ionic concentration of a microdialysis perfusate on basal striatal dopamine levels in awake rats. J Neurochem. 1991,56(2):452-456
69.赵秋华,岳云,于代华,等.氯胺酮和咪唑安定麻醉下脑皮层谷氨酸含量的变化:活体脑微透析研究.临床麻醉学杂志,2001,17(7):381-384
70.伍建林.氯胺酮对鼠纹状体乙酰胆碱释放的影响.中山医科大学学报,1996,17(3):228-230
71.贾兴元,吴安石,岳云,等.微柱高效液相色谱法测定大鼠脑微透析液中的乙酰胆碱和胆碱.色谱,2004,22(1):33-35
72.陈生弟主编,神经变性性疾病.第1版,人民军医出版社,北京.2002:265
73.张兰,李林.线粒体缺陷与阿尔采末病.生理科学进展.1999;30(4):363-366
74.张兰,李林,刘树森,等.叠氮钠对培养神经细胞微管结构和细胞存活率的影响.中国神经科学杂志,2000,16(3):257-262
75.张兰,李林,班立勤,等.叠氮钠对SH-SY5Y人神经母细胞瘤细胞线粒体跨膜电位的影响.中国医学科学院学报.2000;22(5):436-439
76.张兰,叶翠飞,张如意,等.微泵恒速灌注叠氮钠对大鼠学习记忆的影响.中国行为医学科学.2001,10(1):1-3
77.张林,等.微泵恒速灌注叠氮钠对大鼠学习记忆及胆碱乙酰转移酶活性的影响.中国药理学会通讯.2000,17(2):44-45
78.王本祥主编.现代中药药理学.第一版.天津科学技术出版社.天津.1999:1306
79.王巍,等.何首乌及其提取物对小鼠记忆获得障碍的影响.中国中两医结合杂志.1999;19(特):26-28
80.李旻,杜小平,叶晖,等.何首乌对CK所致大鼠脑胆碱能纤维损伤的保护作用.脑与神经疾病杂志.2002,10(3):137-139
81.王巍,赵德忠,孙晓芳,等.中药首茸方对左旋多巴诱发帕金森病大鼠脑内羟自由基增高的影响.中国中西医结合杂志,2003,23(增刊):50-53
82. Lowry OH, et al. Protein mesurement with folin-phenyl agent. J Biol Chem. 1951,193(1):265
83. Huang T, Yang L, Gitzen J et al. Detection of basal acetylcholine in rat brain microdialysate. J Chromatogr B Biomed Appl, 1995,670(2): 323-327.
84. Greaney MD, Marshall DL, Bailey BA, et al. Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor. J Chromatogr, 1993, 622(2): 125-135.
85. Parker WD Jr, Filly CM, Parks JK. Cytochrome oxidase deficiency in Alzheimer's disease. Neurology, 1990, 40(8): 1302-1303.
86. Parker WD, Mahr NJ, Filly CM, et al. Reduced Platelet Cytochrome C oxidase activity in Alzheimer's disease. Neurology, 1994, 44(6): 1086-1090
87. Parker WD Jr, Parks JK. Cytochrome c oxidase in Alzheimer's disease brain: purification and characterization. Neurology, 1995, 45(3 Pt 1): 482-486
88. Mutisya EM, Bowling AC, Beal ME Cortical cytohrome oxidase activity is reduced in Alzhermer's disease. J Neurochem, 1994, 63(6): 2179-2184.
89. Bennett MC, Diamond DM, Stryker SL, et al. Cytochrome oxidase inhibition: a novel animal model of Alzheimer's disease. J Geriatr Psychiatry Neurol. 1992;5(2):93-101
90. Bennett MC, Rose GM. Chronic sodium azide treatment impairs learning of the Morris water maze task. Behav Neural Biol. 1992,58(1):72-75
91.徐海伟,黎海蒂,范晓棠,等.AD大鼠模型的制备及行为学和超微结构研究.中国行为医学科学,2003,12(2):123-127
92. Smith TS, Bennett JP Jr. Mitochondrial toxins in models of neurodegenerative diseases. Ⅰ: In vivo brain hydroxyl radical production during systemic MPTP treatment or following microdialysis infusion of methyllpyridinium or azide ions. Brain Res, 1997, 765(2): 183-188
93.徐海伟,黎海蒂,范晓棠,等.褪黑素在叠氮钠所致的海马锥体细胞钙超载中的神经保护作用.第三军医大学报,2003,25(3):201-204.
94.张兰,张如意,李林,等馓泵恒速灌注叠氮钠致痴呆大鼠脑内胆碱及单胺类神经递质含量改变.中国康复理论与实践,2003,9(9):526-529
95. Nicoletti F, Canonico PL. Glycine potentiates the stimulation of inositol phospholipid hydrolysis by excitatory amino acids in primary cultures of cerebellar neurons. J Neurochem. 1989,53(3):724-727.