Study On Protection Mechanism Of Radix Polygoni Multiflori Preparata In Rats Brain With Sodium Azide Perfusion

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 (NaN₃), as an inhibitor of cytochrome oxidase (COX), can induce the abnormity of oxidative phosphorylation in mitochondria. Animal tests showed that NaN₃ 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 NaN₃ 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 confirmedeffective 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 statisticalsignificance, 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 ofcholinergic 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-Rof 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. Thisindicated 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...