| Background and objectiveThe incidence of vascular dementia is rising for the poplation aging and hign morbidity of cerebrovascular disease.A study's conclusion is that the prevalence of vascular dmentia is 1.1%in over 65-year-old of China's 4 urban region.Explorating vascular dementia risk factors,early diagnosis and therapy have been the emphasis of fundamental and clinical study.Numerous studies have found that amino acid excitotoxicity and apoptosis,at least in part,involved in the pathological process of vascular dementia.Excitatory amino acids contain glutamate and glycine in the brain,glutamate is the largest and the most widely distributed amino acid. Glutamate receptors was divided into metabotropic and ionotropic receptors,ionotropic receptor contains NMDA receptor,AMPA receptor and KA receptor.Hypoxia ischemia will lead to over-activation of the NMDA receptor,resulting in intracellular calcium overload and activation of nitric oxide synthase.Then intracellular calcium overload could cause apoptosis by the depolarization of mitochondria,Cytochrome C release from mitochondria and activation of Caspase family.Memantine is the mild,non-competitive and voltage-dependent NMDA receptor.Memantine have neuroprotective effect on blocking the NMDA receptor,reducing calcium overload and apoptosis.We establisted rat models with vascular dementia to probe the neuroprotective effect mechanism of memantine.The behavioral abnormalities were investigated by Y-type maze test;the changes of cell morphology and the expressions of neuronal nitric oxide synthase(nNOS) and cysteine proteinase-3(Caspase-3) of neuron in hippocampal CA1 region were detected by HE stain and immunohistochemistry respectively;the percentage of neuronal apoptosis by TUNEL.Materials and methods1 Animals and groups 36 healthy male Wistar rats(from Henan Medical Center for experimental animals,months≥18,qualities 400±50 g) were randomly divided into sham-opration group,vascular dementia (VD) group and memantine group(12 rats each group).2 Models and medication Rat models with vescular dementia were establisted by a permanent bilateral common carotid arteries occlusion(2-VO).Then memantine 3mg/kg(dissolved in saline) was given by intragastric administration once a day for 6 weeks; sham-operation group and VD group 1ml saline once a day for 6 weeks.3 Measurement of learning and memory The learning ability was measured by Y-maze test after the oral administration.The criterion is continuously 9 times correct response in 10 times test.The total times of each rat tested is learning records and memory ability test was repeated after 24 hours.4 Specimens The rat brains was obtained by perfusing fixation from heart after the behavior test.The 3mm hippocampus tissues was in turns applicated paraformaldehyde fixation,gradient ethanol dehydration, xylene transparent and wax dip embedment.The 5μm coronal slices of hippocampus were used for HE stains testing morphology change, SP-immunohistochemistry testing nNOS and Caspase-3 and TUNEL stains testing apoptosis.5 Immunohistochemistry and TUNEL stains outcome assessment Caspase-3 and nNOS positive cells were stained brown in cytoplasm.The average gray value were measured by Qwin550CW image processing and analysis system.The percentage of apoptotic cells were taked by adding up TUNEL staining brown positive cells in nucleus.6 Statistical Methods The statistical software SSPS13.0 for windows was exploited to analyis data.All the data were expressed by((?)±s). Deviation and difference were compared using the ANOVA,S-N-K test between each two groups.The difference significance is judged byα=0.05.Results1 Learning and memory performance Compared with VD group and sham-operated group,the memantine group's performance records of learning and memory were in the middle(learning records:63.16±1.75, 79.25±2.01,50.83±1.80,F=707.91,p＜0.01;memory records: 53.17±1.85,69.17±1.70,40.67±1.83,F=762.43,p＜0.01).The three groups' records were significantly different.2 Compared morphological change in hippocampal CA1 region The results after HE staining through light microscope(400 times) were that a large number of normal neurons which lined up orderly,nucleus round or oval,nucleolus clear and chromatin evenly were observed in sham-operation group rats;few normal neurons in VD group rats which structure loosely,nucleus pyknosis and nucleolus vanishing;numerous normal neurons which lined up orderly,chromatin abundant and few nucleual pyknosis in memantine group rats.3 The results of immunohistochemistry Caspase-3 and nNOS positive cells were stained brown in cytoplasmic.Only few weak and moderate stained positive cells were observed in sham-operation group rats through light microscope;a large numbers of deep brown positive cells in VD group rats.The positive cells of memantine group rats were more than sham-operation group rats' but less than VD group rats'.The positive area average integral optical density value in positive area of three groups are significantly different:nNOS average optical density value: 175.87±3.34,140.23±2.30,105.63±3.52,F=1539.67,p＜0.01,Caspase-3 average optical density value:162.78±5.25,132.78±5.36,99.54±5.06, F=439.37,p＜0.01.4 Apoptosis The results were that only little cell apoptosis in sham-operation group,a large number of apoptotic cells in VD group and memantine group's cell apoptosis less than the VD group's but more than sham-operation group's.The percentages of apoptotic cells of three groups were significantly different(33.75±2.70,56.17±3.56, 6.33±3.65,F=672.91,p＜0.01).Conclusion1 Caspase-3,nNOS and apoptosis were involved in the pathogenesis of vascular dementia.2 Memantine could attenuate the hippocampal neurons injury of rat with vascular dementia and improve their learning and memory ability, the mechanism may be through suppressing over-expression nNOS and Caspase-3 and then efficiently reducing neoronal apoptosis. |
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