| Background and ObjectiveVascular dementia(VaD) is a kind of acquired intelligence damage syndrome caused by brain dysfunction resulted from various kind of cardiovascular disease, it was a chronic progressive disease. Cognitive impairment,decrease the ability of memory,and change of the psychological signs were Its main clinical manifestation, which has not been effectively dealed with by now.With the deep studies on Vascular dementia, some scholar believe that N-methy1-D-aspartate (NMDA) receptor connect with the cognitive impairment in Vascular dementia. N-methy1-D-aspartate receptor was a isomerism pentamer that made up the subunit proteins, different the subunit proteins showed different characteristic of the physiology and pharmacology. NR1 and NR2B were main subunit proteins in hippocampus area. NR1 was the cardinal function subunit proteins for NMDA receptor, calcium ion(Ca2+) aisle was mainly regulated by it. With the increas of calcium ion level, endonuclease was activited and DNA(deoxyribonucleic acid) was degradated, apoptosis was started, those induced the death of the hippocampus neuron. While NR2B was played important role in the synaptic plasticity and learning and memory.There was a great deal of free radical in vascular dementia, especially hydroxy radical, its toxicity was very strong to the cells, while glutathion peroxidase(GSH-PX) and catalase(CAT) are two kind of main enzyme in decomposed hydrogen dioxide(H2O2), they were played important action in cleaning of the free radical in organism.It has been reported recently that cholinesterase inhaibitor(CHEI) could protect against ischemia, such as huperzine A. Donepezil is a potent and selective acetylcholinesterase (AChE) inhibitor developed from the treatment of Alzheimer's disease. Recent years, it was used to the Vascular dementia. Whether existe the other action mechanism or not has been reported rarely at present. The experiment aimed to probe the effection of Donepezil on expression of N-methy1-D-aspartate receptor subunit proteins NR1,NR2B and activity of GSH-PX,CAT on Neuron of Hippocampus in Mice with Vascular Dementia. All of this will be provided some consummate theory evidences for the clinical use of Donepezil.Materials and methods(1) The rats were subjected for cerebral ischemia-reperfusion repeatedly on bio-lateral common carotid arteries occlusion and injected sodium nitroprusside into the abdominal cavity to establish the imitative vascular dementia rat models.(2) 48 male Sprague-Dawley rats each weight 400±50g were randomly divided into three groups(sixteen rats each group).Group A: sham operation group; Group B: model group and Group C: donepezil group. Group C was received a treatment of donepezil(1mg /kg /d), treated for 30d serially. At the same time group A and B were poured into the stomach with saline for 30d serially altogether.(3) Ability of learning and memory of each group of rats were observed by Y-type maze test by means of detection of total refection time (TRT )and error number (EN); The activity of GSH-PX and CAT were measured with dithio-bis-nitrobenzoic acid (DTNB) and ultraviolet spectrophotometry(UV) methods respectively.(4) Section of every mices was processed with HE and Nissl staining, the expressions of N-methy1-D-aspartate receptor subunit proteins NR1 and NR2B in neuron of hippocampus were detected by immunohistochemistry technique. The average number of positive cells in every section was calculated with Picture Analysis Software. (5) All data was dealed with SPSS11.0 statistic software. All the data wereexpressed with Mean±St.Deviation( x±s), and analyzed with One-Way analysis ofvariance among group, and compared with S-N-K and LSD method between two group. The significant testing standard was a= 0.05Results1. Test of learning and memory:1.1. After VaD model made 7 days, the result of group C was better than group B but lower than group A, group B was much lower than group A (P<0.01). These result indicated that imitative vascular dementia rat models was success.1.2. After VaD model made 25 days, the result of group B was much lower than group A and C (P<0.01) , while between group A and C were no significant (P>0.05).2. Activity of GSH-PX:Compared with group B (57.35±14.84), the activity of group C (82.81±9.14) was increased evidently (P<0.05) , there was no obvious difference of the results between group C and group A (98.67±13.85) (P>0.05)3. Activity of CAT:Group B (14.32±3.87) was decreased intensively than group A (31.91±6.54) (P<0.01) and C (28.19±6.32)(P<0.05), but no significant was found between group C and A(P>0.05).4. The pathological changes:4.1 HE staining in every group (×400 )Group A: the layer of structure was close and clear, neuron in morphology in hippocampusCA1 area was not found abnomal under light micoroscope; Group B: normal neuron reduced obviously, neuron arranged very sparse and chaotic, the layer of structure was very vague and the cell interval becamed widen, many nucleus shrinked firmly and nucleolus disappeared, the section appeared a lot of irregular gliocyte and gliocyte hyperplastic knot; Compared with group B, group C 's section showed that normal neuron increaded evidencely, cell arranged closer and nucleolus was clearer, only a few degenerative and shrinking neurons were found. 4.2 Nissl staining in every group4.2.1. Light micoroscope(×400) Group A: neuron and neurapophysis were very clear and complete; Group B: a lot of shrinking and deformed neuron was found all of the visual field under the micoroscope; On the contrary, group C can be found many distinct and intact neuron or neurapophysis.4.2.2. Oil micoroscope(×1000) Group A can be found that patching shaped Nissl body was very plentiful, nucleolus was clear very much, between nucleus and endochylema can be very easily distinguished; Group B can be seen that a great many Nissl body reduced or disappeared, between nucleus and endochylema couldn't be discerned; But diffent from group B, group C spot film shaped Nissl body had been improved instently, which becomed much more than group B, between endochylema and nucleus were easily differentiated.5. The expression of NR1 and NR2B5.1. Distribution of NR1 in every group (SP×400)Group A: the layer of structure was extreme close and clear in hippocampusCA1 area, cell membrane of positive neuron of NR1 was showed yellow and endochylema was lighter; Group B: the layer of structure was lessen and loosen, but cell membrane of remaining positive neuron of NR1 was appeared deep Buffy, some colorized nucleus can be found; There was difference form group B, the quantity of neuron was obviously improved in group C, the layer of structure was also get clearer and closer, but expression of NR1 was significantly decrease.5.2. Distribution of NR2B in every group (SP×400)Group A: a great many positive neuron of NR2B was expressed in hippocampusCA1 area, cell membrane was colorized deep Buffy and endochylema was lighter; Group B: the layer of structure was unclear and disorder, the expresse of NR2B was sharply reduced, only a few lighter positive neuron of NR2B was expressed; On the contrary, compared with group B, the expresse of NR2B in group C was evidently increased, clearer and closer can be seen in sections in hippocampusCA1 area.5.3. Comparison of NR1 gray scale value in every group Group A was 38.78±1.06, group B 57.94±2.56 and group C was 35.18±1.94, the resoult showed that group B was heighten significantly compared with group C and A(P<0.01), but there was no obvious difference between group C and A(P>0.05).5.4. Comparison of NR2B gray scale value in every groupGroup A was 58.36±1.55, group B 27.69±1.97 and group C was 50.87±1.50, the resoult showed that group B was lower sharply compared with group C and A(P<0.01), no significant was found between group C and A (P>0.05).Conclusion(1 ) Over expression of NR1 and sharp reduction of NR2B in hippocampusCA1 area, and the decreased activity of GSH-PX and CAT were related with vascular dementia;(2) Donepezil can protect the neuron of hippocampus in mice with vascular dementia, which neuroprotective mechanism may be connect with lower expression of N-methy1-D-aspartate receptor subunit proteins NR1 and high activity of GSH-PX and CAT.(3) Donepezil may not only reduce cognitive impairment by improving cholinergic function in the brain, but also protect neuron of hippocampus to avoid injury and increase the expression of NR2B by decreasing the expression of NR1 and improving morphological structure in vascular dementia. |
