1 褪黑素和银杏叶提取物 对大鼠记忆的影响

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infusedintracerebroventricularly with h -amyloid 1–40F.Tang *,S.Nag 1,S.Y .W.Shiu,S.F.Pang 2Department of Physiology,Faculty of Medicine,The University of Hong Kong,Hong Kong,ChinaReceived 11March 2002;accepted 3June 2002AbstractIntraventricular infusion of rats with h -amyloid for 14days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus.These changes were not altered by daily intraperitoneal injection of 20mg/Kg melatonin.Orally administered Ginkgo biloba extract,however,partially reversed the memory deficit and the decrease in choline acetyltransferase activities in the hippocampus.The latter treatment failed to reverse the decrease in somatostatin levels.The results indicate that orally administered Ginkgo biloba extract can protect the brain against h -amyloid-induced changes which lead to memory deficit through its effect on the cholinergic system.D 2002Elsevier Science Inc.All rights reserved.Keywords:Cholinergic;Ginkgo biloba;melatonin;somatostatin;water-mazeIntroductionIn our previous studies,intracerebroventricular (i.c.v.)infusion of rats with h -amyloid for 14days results in memory deficit in the Morris water maze as well as decreases in choline acetyltransferase0024-3205/02/$-see front matter D 2002Elsevier Science Inc.All rights reserved.PII:S 0024-3205(02)02105-7*Corresponding author.Tel.:+852-28199-269;fax:+852-28559-730.E-mail address:ftang@hkucc.hku.hk (F.Tang).1Present address:Department of Anatomy and Physiology,Meharry Medical College,1005D.B.Todd Boulevard,Nashville,TN-37208,USA.2Present address:CK Life Sciences International Inc.,2Dai Fu Street,Tai Po Industrial Estate,New Territories,Hong Kong,China./locate/lifescieLife Sciences 71(2002)2625–2631(ChAT)and somatostatin (SRIF)levels in the cerebral cortex and hippocampus [1,2].As an anti-oxidant[3],melationin has been shown to protect against hippocampal damage by kianic acid [4]and quinolinic acid [5]by preventing the formation of lipid peroxidation products.Melatonin and its related compounds have also been shown to protect neurons against the toxic effects of h -amyloid [6].Furthermore,it has been postulated that the loss of intraventricular fluid melatonin may lead to the neuropathology of Alzheimer’s disease [7].Similarly Ginkgo biloba extract EGb761has been shown to protect neurons from beta amyloid toxicity [8]and to enhance short-term memory in both young and aged rats [9].In addition,Gingko biloba has been shown to improve cognitive functions in Alzheimer patients [10].It is therefore of interest to look at the effects of melatonin and Ginkgo biloba extract in our h -amyloid infused rat model.Materials and MethodsMale Sprague-Dawley rats (3months of age,weighing 250–300gm)were obtained from the Laboratory Animal Unit of the University of Hong Kong.They were kept in a temperature controlled room (22j C)under a 12L:12D cycle (light on at 600h)and were given free access to food and water.In the first experiment,there were 3groups:(1)vehicle infused and vehicle injected (veh +veh),(2)h -amyloid infused and vehicle injected (Abeta +veh)and (3)h -amyloid infused plus daily i.p.injection of 20mg/Kg melatonin (Abeta +Mel).In the second experiment,there were 4groups:(1)vehicle infused plus oral vehicle (veh +veh),(2)vehicle infused plus oral Ginkgo biloba extract,100mg/Kg (veh +Egb),(3)h -amyloid infused plus oral vehicle (Abeta +veh),and (4)h -amyloid infused plus oral Egb,100mg/Kg (Abeta +Egb).The Ginkgo biloba extract was a generous gift from CV Technologies,Canada and contained 24%flavoid glycosides and 6%terpenoids.In both experiments,h -amyloid infusion were continued for 14days and melatonin or Ginkgo biloba extract was also given daily in the afternoon throughout the same period.The preparation of h -amyloid and surgery for continual i.c.v.infusion of h -amyloid by Alzet mini osmotic pumps (Alza Corporation,Palo Alto,CA,U.S.A.)have been described [1].Oral administration of Ginkgo biloba extract (50mg in 1ml water )in suspension was by the use of a stomach tube.The water maze tasks for spatial reference memory has also been described [1,2].It lasted for 5consecutive days,starting on postoperative day 10.One block of visible platform trials and one block of acquisition trials (with the platform submerged in milky water)were administered on postoperative day 10.Two blocks each of acquisition trials were given on days 11–13.Escape latency (the time the rat took to swim to the platform)was measured.On day 14the rats were given one probe trial in which the time spent in the target quadrant during a 60second swimming period was noted.The rats were decapitated on post-operative day 15and their cortices and hippocampi dissected out on ice to be stored at À70j C.Choline acetyltransferase (ChAT)activities were measured by a radioenzymatic assay [2]and somatostatin levels determined by a radio-immunoassay [11]using an anti-serum generously supplied by Dr.J.Hong (Research Triangle Park,NC,U.S.A.).The protein contents were estimated using the protein reagents from Biorad (Hercules,CA,U.S.A.).The data of the first experiment were subjected to one-way analysis of variance (ANOV A)while the two-way ANOV A was used for those of second experiments.These were followed by a post hoc LSD test for differences between groups.The statistical significance level was set at p <0.05.F .Tang et al./Life Sciences 71(2002)2625–26312626ResultsIn the first experiment (i.e.i.p.melatonin,Fig.1),ANOV A of the mean escape latencies per 4-trial block demonstrated significant effects of Group [(F(2,21)=7.639,p <0.001],and Blocks [F(6,126)=10.316,p <0.01]and LSD post hoc test revealed that veh+veh group significantly differed from the Abeta+veh and Abeta+Mel groups (p <0.01).Abeta+veh group was not different from Abeta+Mel group.In the probe test,there was a significant effect of group [F(2,23)=6.505,p <0.01].The LSD post hoc test showed significant differences between veh +veh and Abeta +veh or Abeta +Mel groups.Abeta +veh was not different from Abeta +Mel group.The ChAT values for the cortex in the veh +veh,Abeta +veh and Abeta +Mel groups were 0.86F 0.07,0.54F 0.06and 0.64F 0.10respectively,and for the hippocampus were 0.64F 0.04,0.41F 0.02and 0.33F 0.03respectively.Similarly,the SRIF values for the cortex in the veh +veh,Abeta +veh and Abeta +Mel groups were 4.42F 0.68,1.87F 0.24and 1.96F 0.39respectively,and for the hippocampus were2.30F 0.41,1.07F 0.33and 1.29F 0.23respectively.h -Amyloid infusion led to significant reduction in ChAT [F(2,24=3.589,p <0.05for cortex;F(2,24)=2.26,p <0.01for hippocampus]and in SRIF [F(2,24)=9.633,p <0.01for cortex;F(2,24)=3.881,p <0.05for hippocampus).The LSD post hoc test showed that veh +veh group significantly differed from the other 2groups (p <0.05).Again Abeta +veh did not not differ from Abeta +Mel group.In the experiment with Ginkgo biloba extract (Fig.2),the performance of the Abeta +veh group was selectively impaired in the acquisiton phase.A 2Â2Â7(Abeta ÂEgb ÂBlocks of 4trials)split-plot ANOV A of the escape latency revealed a significant effect of Blocks [F(6,168)=38.14,p <0.01]and of Abeta [F(1,28)=29.31,p <0.01].Egb had no effect.However,there was a significant interaction between Abeta and Egb [F(1.28)=13,42,p <0.01]suggesting a restoration of performance in Abeta +Egb group as indicated by their lower escape latencies compared to Abeta +veh group.Probe test analysis further supported the above interpretation.The memory of the Abeta +veh group was selectively impaired (spent less time in the target quadrant)which can be seen as a significant effect of Abeta [F(1,32)=17.44,p <0.01].Egb alone had no effect whereas there was a significantinteraction Fig.1.Water-maze performance from Experiment 1.(A)Mean escape latency for the visible platform task,expressed as a function of trials.(B)Mean escape latency for the hidden platform task,expressed as a function of blocks of four trials.(C)Performance in the probe test,expressed as time spent in the target quadrant.F .Tang et al./Life Sciences 71(2002)2625–26312627between these two factor [F(1,32)=7.71,p =001).This interaction suggests that Egb restored the memory of Abeta-treated animals so that they spent more time searching for the platform in the appropriate quadrant.Again h -amyloid infusion led to a significant reduction in ChAT [F(1,32)=45.11,p <0.01for cortex and F(1,32)=28.13,p <0.01for hippocampus]and in SRIF [F(1,32)=18.82,p <0.01for the cortex and F(1,32)=35.78,p <0.01for hippocampus](Table 1).Egb had significant effect on ChAT in the cortex [F(1,32)=6.23,p =0.019]and in the hippocampus [F(1,32)=4.58,p =0.041].There was also a significant interaction between Abeta and Egb in hipocampal ChAT (F(1.32)=7.19,p =0.012],showing that Egb significantly increased ChAT level or prevented them from declining in the Abeta treated-rats.The LSD post hoc test showed that Abeta +veh group significantly differed from veh +veh and veh +Egb groups (p <0.005]in both ChAT and SRIF.The LSD test also showed that ChAT in the hippocampus in Abeta +Egb group was significantly higher than Abeta +veh (p =0.002)although it was still lower than in veh +veh (p =0.033).As to the ChAT in the cortex,the only effect of Egb was to increase it in veh +Egb group compared with veh +veh group (p =0.035).Egb had no effect on SRIF.Table 1Neurochemical measurements in cerebral cortex and hippocampus of i.c.v.Abeta/veh +oral Egb/veh treated ratsveh +vehAbeta +veh veh +Egb Abeta +Egb ChAT (U/mg protein)Cortex1.27F 0.100.68F 0.06 1.57F 0.120.86F 0.09Hippocampus0.48F 0.020.33F 0.020.47F 0.020.42F 0.02SRIF (ng/mg protein)Cortex2.87F 0.50 1.46F 0.20 2.80F 0.26 1.51F 0.17Hippocampus3.32F 0.37 1.27F 0.27 2.65F 0.25 1.39F 0.18Data are presented as mean F S.E.M.n =8for allgroups.Fig.2.Water-maze performance from experiment 2.See Fig.1for details.Significant difference between Abeta +Egb and Abeta +veh groups emerged from 4th Block onward.F .Tang et al./Life Sciences 71(2002)2625–26312628F.Tang et al./Life Sciences71(2002)2625–26312629 DiscussionThe result of this study indicates that i.p.melatonin had no effect while orally given Ginkgo biloba extract restored memory to some degree and increased ChAT activities towards normal only in the hippocampus.Preliminary data from i.c.v.administered melatonin(20A g/kg B.W)are not different from those obtained by i.p.injection.From the results of oral Ginkgo biloba extract on ChAT,one may conclude that the cholinergic system in the hippocampus is more important than that in the cortex for memory,at least in the Morris water-maze system,which is insensitive to lesions of the entorhinal cortex [12].This is in line with the current understanding of the importance of the hippocampus in memory function[13].Whether the effect was mediated by an increase in cholinergic activities in the existing fibres or by an increase in the number of cholinergic fibres requires further study.What are the mechanisms behind the effects of oral Ginkgo biolba extract on ChAT and memory loss? Ginkgo biloba extract could have protected the cholinergic neurons against h-amyloid toxicity through its anti-oxidant effects[14].Furthermore,Ginkgo biloba extract could enhance memory through an increase in cholinergic[15]and serotonergic[14]neurotransmission and increase in brain circulation. The negative results obtained with i.p.and i.c.v.given melatonin were unexpected because melatonin has been shown to have anti-oxidant effect[3]and to protect neural tissues against h-amyloid toxicity in vitro[6].Melatonin MT2(Mel1b)receptors has also been found in the hippocampus[16].This discrepancy may be because the anti-oxidant effect of melatonin may not be the only major factor involved and because in vitro and in vivo situations are different.In this connection,it is pertinent to note that Ginkgo biloba has been shown to have in vivo neuromodulatory effects by increasing the gene expression of a number of proteins associated with brain function[17].The lack of effect of oral Ginkgo biloba extract on somatostatin levels may explain why the memory deficit was not completely reversed as somatostatin has been suggested to play some role in memory [18,19].Alternatively,it could be just because the ChAT activities(an indicator of cholinergic activities) in the hippocampus were not completely restored to normal.The significance of the cholinergic system in memory is well-established[12].The result here cannot resolve this point.The beneficial effects of Gingko biloba extract on the memory of h-amyloid infused rats are in line with some of the findings in clinical trials which show a positive effect of Gingko extract on improvement of congitive functions[20,21].These positive clinical findings,however,have not been confirmed by a later study by van Dongen et al.[22].Although the results of this study do raise a number of issues,it leaves little doubt as to the beneficial effects of oral Ginkgo biloba extract on the cholinergic system and memory loss in our rat model.We must,however,emphasize that the h-amyloid infused rat model is at best a partial model of Alzheimer’s disease.There are quite a few intrinsic differences.Firstly,the time frames are different because our model is based on a short time of infusion(i.e.14days),which is not long enough for the development of neuronal loss or neural degeneration,a characteristic feature in Alzheimer’s disease. The second limitation is that the increase in h-amyloid may not be the only factor involved in the development of Alzheimer’s disease although it is likely to be the key factor.The discrepancy between this study and some of the clinical trial with negative results may be explained by these differences and also by the much higher dose of the Gingko extract used in the rat model than in the clinical trials (some twenty to fifty times higher in the rat).Regardless of the limitations stated above,the rat model may still be useful for the study of Alzheimer’s disease especially if the duration of h-amyloid infusion can be increased.AcknowledgementsWe are grateful to the Executive Panel for the Review of the Preclinical Departments,the University of Hong Kong and the Vice-Chancellor Development Fund for Traditional Chinese Medicine,for their financial support,Dr.John Hong,Research Triangle Park,NC,U.S.A.for his generous gift of somatostatin anti-serum,the CV Technologies,Canada,for the gift 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