1.A Comparative Study With Clinical Comprehensive Assessment For Mild Cognitive Impairment And Alzheimer's Disease 2.Experimental Research Of Molecular Biology Mechanism And Intervention On Synapse Functional Disturbance Of The Brain In The Alzheimer'

Objective:To further clarify its possible mechanism and provide the theory basis for the prevention and cure of MCI and to elevate specificity and sensibility for early diagnosis of AD,we examined the changes of rCBF,P300,MMSE and CSF biological markers such as GAP-43,Aβ1-42 in patients with MCI,AD and CN.Methods:Totally 25 patients with Alzheimer's Disease(AD),36 patients without Mild Cognitive Impairment(MCI)and 30 Cognitive Normal(CN)cases,who came from the Department of Neurology,Second Affiliated Hospital of Chongqing University of Medical Sciences from January 2006 to December 2007,were enrolled in the study. The diagnosis of probable AD was made according to criteria proposed by the National Institute of Neurological and Communicative Disorders and Stroke--Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) and the diagnosis of MCI was made by Petersen's criteria.The CN group was consisted of patients with other complaints such as back pain and headache without cognitive impairment. All patients and controls were subject to the analysis with individual and (or) legal guardian consent for the study.MMSE tests was performed on all participants to confirm their cognitive status.3ml of CSF was obtained by lumbar puncture from individuals; then CSF were stored at -70°C until needed.P300 was measured with auditory sense oddball program of myoelectricity-evoked potential apparatus.SPECT rCBF imaging was done and the characteristics of rCBF were analyzed by semi-quantity technique.To measure the quality concentrations of GAP-43,Aβ1-42 in CSF by Enzyme-linked Immunoabsorbent Assay technology.Results:(1)The three groups were proved matched in sex,age and educational degrees. MMSE tests in AD group were significantly 1ower than those in the CN and MCI group.Compared with CN group,MCI group achieved obviously 1ower scores in MMSE tests.(2)The P300 latency in both AD and MCI group was significantly delayed than that of the CN group to different degrees.The amplitude of P300 were lower in the AD group, which was significantly different from that in the CN group and the MCI group.There was no significant difference in the P300 Amplitude between the MCI group and the CN group.(3)The RAR of bilateral frontal,parietal,temporal lobe,hippocampus,cingulate gyri and left thalamus in AD group was significantly lower than that of CN group to different degrees.The RAR of bilateral temporal lobe,left hippocampus and cingulate gyri in MCI group was significantly lower than that of CN group to different degrees,especially in the cingulate gyri.(4)The CSF level of GAP-43 of AD group was obviously higher than that of MCI group and CN group,but there was no significant difference between MCI group and CN group.The CSF level of Aβ1-42 of AD group and MCI group was significantly lower to different degrees than that of the CN group,especially in AD group.Conclusion:(1)The experimental results indicated that significant hypoperfusion in bilateral temporal lobe,left hippocampus and cingulate gyri may be useful in predicting who will convert from CN to MCI . It may reflect dynamic change of rCBF and the severity of cognition impairment among MCI,AD and CN group to a certain extent.(2)To monitor the cognitive dysfunction,the extension of P300 latency is earlier than the depression of P300 amplitude. The finding indicates that studies of P300 latency would be useful in early diagnosis of AD and in the observation during the treatment(.3)The CSF levels of Aβ1-42,GAP-43 may be useful as the biomarkers of AD,not only for the very early specific diagnosis,but also for the monitoring the therapeutic effect.(4)It is useful for detecting the MCI and AD as well as monitoring the severity of AD to carry out the MMSE,rCBF,P300 and the CSF levels of Aβ1-42,GAP-43 testing.The results also reveal that the interplay of different markers might be considered for enhancing diagnostic accuracy of pre-clinical AD. Objective: The present study was carried out to investigate the effect of DiHuang YiZhi JinGao(DHYZJG)on the learning-memory ability,the function of central cholinergic system,molecular and cellular signal transduction pathways,synaptic plasticity in the rat model of Alzheimer's disease(AD) so as to explore the possible mechanism and provide the experimental evidence on clinical application of DHYZJG in treatment of AD.Methods:(1)The DHYZJG produced by Institute of Chinese Materia Medica,ChongQing was composed of 8 Chinese herbs such as prepared rehmannia root,medicinal cornel, acori graminei,rhizoma, Polygala,morinda root and so on. (1g DHYZJG is equivalent to 2g crude drug). To study the safety and toxic Reactions of DHYZJG in acute toxicologic experiment in mouse.(2)Health male 90 Sprague-Dawley (S-D)rats were randomly divided into 6 groups. Aggregated Aβ(25-35)was injected into the right hippocampus of the rats to make experimental rat model of AD. The behavioral abnormalities were investigated by shuttle box and the water maze performance.(3)The histopathologic changes of right hippocampus CA1 region of rats were observed by HE staining and nissl staining.(4)Ach content and AchE activity were measured by the modified alkaline hydroxylamine colorimetric method and the improved Ellman assay, respectively. The expression of mRNAs related to M-AchR M1,N-AchR a4,a7 were observed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Western-blot was used to analyze the expression of CREB,pCREB Ser133,GSK-3βand pGSK-3βSer9 in hippocampus of the rats. The effects of DHYZJG on AD rat model were studied by using Donepezil as control group(.5)The protein expression of GAP-43,PSD-95 of right hippocampus CA1 region of rats was estimated with immunofluorescence technique and the confocal laser scanning microscopy system.The impairment on the structure of synaptic interface in right hippocampus CA1 region of rats were quantitatively analyzed by electron microscopy and computer image processing appliance (①Numerical density(Nv);②the thickness of the postsynaptic densities (PSD);③the length of the active zones and④the curvature of the synaptic interface). The effects of DHYZJG on neuronal synaptic plasticity in the hippocampus region of AD rats were studied. Result:(1)After 4 weeks treatment: the ability of learning and memory of the Model group was significantly decreased. DHYZJG can markedly improve the function of learning and memory of in AD rats in a dose dependent manner. ( 2 ) After 4 weeks treatment:the pathomorphological examination as HE staining, Nissl staining showed that in the Model group the impairment as the severe neuron damage and loss,chromatin condensation,nuclear karyopyknosis and pyknosis,degradation of nissle body.The neurons in right hippocampal CA1 area in the DHYZJG Medium,High group showed less severe damages than that in the Model group,seldom seeing apoptosis.Adopting Nissl staining,analyzing by image acquiring and analysis system of computer, the number of neurons in right hippocampal CA1 area in the Model group decreased significantly compared with the Sham group, while the degree of neuron loss in DHYZJG Medium,High group was relieved than that in the Model group.(3)After 4 weeks treatment:compared with the Sham group,the right hippocampus of the Model group showed significantly diminished expression of Ach content,AchE activity. The contents of ACh and AChE activity in right hippocampus were obviously increased to different degrees in the DHYZJG Medium,High group compared with the Model group.Such promotion was dose-dependent.(4)After 4 weeks treatment: compared with the Sham group,the right hippocampus of the Model group showed diminished expression of pCREB Ser133 and pGSK-3βSer9, while the level of CREB and GSK-3βwas no significant difference.Compared with the Model group,the contents of pCREB Ser133 and pGSK-3βSer9 in right hippocampus of the DHYZJG High group were significantly increased to different degrees, while the level of GSK-3βwas obviously decreased. (5)After 4 weeks treatment:compared with the Sham group,the expression of N-AchR a7mRNA in right hippocampus of the Model group were obviously increased,while the level of M-AchR M1,N-AchR a4 mRNA was no significant difference. Compared with the Model group,the mRNA expressions of M-AchR M1,N-AchR a7 in right hippocampus of the DHYZJG High group were obviously increased.(6)After 4 weeks treatment: compared with the Sham group,in right hippocampus CA1 zone of the Model group the Nv,the thickness of PSD,the length of the active zones of the synapses were significantly decreased, the expression of GAP-43 was increased remarkably,while the level of PSD-95 was significantly decreased.The Nv,the thickness of the PSD,the length of the active zones of the synapses and the expression of GAP-43 and PSD-95 in right hippocampus CA1 zone of the DHYZJG High group was significantly increased to those in the Model group.No significant change of the curvature of the synaptic interface among the four group was found.Conclusion:(1)DHYZJG can improve dysfunction of learning and memory in AD rats in a dose-dependent manner(.2)DHYZJG can alleviate the dgrees of cellular necrosis and apoptosis on hippocampus of AD rats to educe protective effect in a dose-dependent manner.(3)These results indicate in AD rats the cholinergic nervous system is severely damaged. DHYZJG can improve central cholinergic dysfunction in AD rats in a dose-dependent manner to attain a new dynamic balance in the modulation of central cholinergic system. The neuroprotective effects of DHYZJG on AD are probably exerted via a multi-target, adjustment in the whole mechanism.(4)These results indicate in the hippocampus of AD rats there was synaptic disconnection and the information transfer,processing and save was obstructed among neuron.DHYZJG may promote synapse reconstitution, enhance synaptic plasticity,improve the axonal growth of damaged neuron and functional reconstruction of central nervous system.