| [Objective] Neuroremodeling is an important mechanism of the occurrence and maintenance in VD(Vascular Dementia),including the reduction of neurons ? the proliferation of glial cells ? the remodeling of dendrites and neurites ? the disordered connection and degeneration of synaptic.This study researches on neurons apoptosis and inflammation interfered by MDPs(Modified Dioscorea Pills)in hippocampus of VD rats to explore the protection function in hippocampal pyramidal cells and neuroremodeling prevention by MDPs;Secondly,it researches on the proliferation of hippocampal Astrocytes and oligodendrocytes and regeneration of nerve fibers interfered by MDPs in hippocampus of VD rats to explore its intervention mechanism in glial network remodeling;Thirdly it researches on the function of synapses protection and synaptic regeneration interfered by MDPs in hippocampus of VD rats to explore its effect on enhancing neuron interaction and ensuring correct conduction of nerve information.Through three aspects of prevention neuroremodeling to further explore the mechanism of treatment and improve intelligence of MDPs in VD.[Methods] 40 SPF male rats were randomly divided into sham operation group,model group and drug-treated group.In the model group and the drug group,the VD models were established by the modified 2-vessels occlusion(2-VO,the left Common Carotid Artery and the right CommonCarotid Artery)method.Rats in the sham operation group were operated in the same way as the operation group except not undergoing 2-VO.After a week of the operation,rats in the drug-treated group were intragastric administered with MDPs oral liquid(prepared by the Pharmaceutical Preparation Room of Hubei Provincial Traditional Chinese Medical Hospital.The dosage is 1 ml /100 g body weight / day)for 45 days,Rats in the other two groups were intragastric administered with normal saline which dose and treatment course were the same with the drug group.Following the lavaging treatment,the capacity of learning and memory of rats in the three groups was evaluated by morris water maze(MWM)behavioral test(including navigation experiment and space exploration experiment).At the end of MWM test,the hippocampus of rats was harvested which included fresh sampling and perfusion sampling.The former is for electronic microscopy and RT-PCR(real-time quantitative polymerase-chain reaction)detection whereas the later is for HE staining and immunohistochemical staining.In HE staining observation,the CA1 region in hippocampus tissue of rats is focused on,including the changes in pyramidal cells layer,lacunar layer,radiation layer and polymorphic layer;In immunohistochemical detection,the targets of Bax,Bcl-2 and NF-K B were analyzed to explore the neurons apoptosis and inflammation regulation of rat hippocampus in three groups to reflect the nerve protection and neuroremodeling;Adopt the targets of Olig-2(Oligodendrocyte transcription factor 2)and GFAP(glial fibrillary acidic protein)to analyze the proliferation of oligodendrocytes and astrocytes to reflect hippocampal neural glial remodeling;Adopt the targets of SYP(Synaptophysin),PSD-95(postsynaptic density protein 95),MAP-2(Microtubule associated protein-2)which represent the synaptic protein to understand the destruction and regeneration of synapse and reconnection between synapses;RT-PCR was used to analyze the geneexpression of GAP-43(growth-associated protein-43)mRNA in the hippocampus to further study the regulation of nerve regeneration and synaptic reconstruction;With transmission electron microscope(TEM)the ultrastructural changes ? reconstruction and regeneration of synapse were recorded.On the mean time,the body weight among pre-operation,after-operation and before sampling were recorded and the mental state,diet and water intake were observed.[results] The general state is as follows: After the modified 2-VO operation,the amount of water intake and diet of rats in the 2-VO group significantly decreased,the spirit and activity degree were low,drowsiness was common in the 2-VO group.After a week of operation the mean weight of rats in the 2-VO group was significantly less than it in the sham-operated group(F-value:19.535,P-value:0.000).Following about two weeks of rehabilitation and feeding,rats in the 2-VO group recovered fast,eating much more food and drinking much more water than the rats in sham operation group.After 50 days of feeding,on the day of sampling there is no significant difference in body weight among three groups(F-measure: 0.010,P:0.990).In the navigation test of morris water maze experiment,there was no statistical difference in escaping latency and escaping distance among three groups in the first two days.But from the third day,the escaping distance and escaping latency of rats in the sham operation group decreased more significantly compared with the rats in the model group and the comparison reached a significant difference(P<0.05).On the fourth day,the escaping distance and escaping latency of the drug-treated group decreased rapidly,which were close to those of the sham operation group and these two indexes of the sham operation group and drug-treated group were much lower than those of the model group and the comparisons reached significant differences(P<0.05,P<0.05).In the spatial probe test,compared to the rats inthe model group,rats in sham operation group and drug group cross over the platform more times and the comparison reached significant differences(P<0.01 and P<0.05).the time and the distance swimming in the platform quadrant of rats in sham operation group and drug group were much more than those of rats in the model group and the comparisons had significant differences(P<0.05 and P<0.01).There was no significant difference among three groups in total swimming distance and swimming speed.In HE staining observation: in the sham operation group,the pyramidal cells in the hippocampal CA1 area of the sham operation group are well arranged in 3-5 layer,the cells are neat and tidy,the cytoplasm is homogeneously dyed-red,the nucleus is large and round,and the nucleolus is clear.The radiation layer is arranged in a regular radial way,and the glial cells are few and the oligodendrocytes with round nucleus can be seen.The cells in the molecular layer arrange densely,the intercellular space is less.The cells number of hippocampal CA1 region in the model group decrease very much,the cells are sparsely and disorderly arranged,the pyramidal cells line in 1-3layer with intact morphology,many cytoplasmic vacuoles,hyperchromatic and pyknosis nuclei with triangular or irregular shape,some vague or even disappeared nucleolus.The radiation layer is loose,the polarity of cells is disorder which extend to the molecular layer and lacunar layer.In the Lacunar layer the fibers are disorientated with many large gaps.Glial cell proliferation is visible.In the drug-treated group,the neurons in hippocampal CA1 area are orderly arranged in 5-7 layers,the pyramidal cells proliferate obviously.The radiation layer is dense and arranges regularly.In the molecular layer and lacunar layer a large number of pyramidal cells and glial cell emerge and proliferates.The expression of GAP-43 mRNA in RT-PCR test: in the sham operationgroup the expression of GAP-43 mRNA is lower than that in the model group and the comparison is significant(P<0.01).However,The expression of GAP-43 mRNA in the drug group is even higher than that of sham operation group and model group and the comparisons reach significant differences(P<0.01,P<0.05).The results of immunohistochemistry is respectively as follows:1 apoptosis and inflammatory index: the expression of Bax: Among three groups,the expression of Bax in the model group is the highest,when compared to the counterpart in other two groups,the differences are significant(P < 0.01,P < 0.01);the expression of Bax in the sham operation group is the lowest,compared with that of the drug group it reached a very significant difference(P < 0.01);The expression of Bcl-2: the expression of Bcl-2 in drug group is the highest,which is significantly higher than that in sham operation group and model group and the comparisons are very significant(P < 0.01,P < 0.01).The expression of Bcl-2 in the sham operation group is significantly higher than it in the model group(P < 0.01);The expressions of NF-B: The expression of NF-B in the drug-treated group and the sham operation group are much lower than it in the model group and the comparison reaches a very significant difference(P?0.01).The expression of NF-B in the drug group is higher than it in the sham operation group and the comparison reaches a significant difference(P?0.05).2 Index of neural glial cells proliferation: Olig-2 expression: the expression of Olig-2 in sham operation group is highest among three groups,the counterpart in the drug group is second.Comparisons of the expression of Olig-2 in the former two groups to it in the model group reach extremely significant differences(P < 0.01,P < 0.01);When the expression of Olig-2 in the drug-treated group compared with it in the sham operation group,the result get a significantly difference(P < 0.05).Whereas in the model group,itexpressed the lowest;The expression of GFAP: The expression of GFAP in the sham operation group is the lowest among three groups,it get very significant differences(P < 0.01,P < 0.01)when compared with it in the other two groups.Comparison the expression of GFAP in the drug group with it in the model group reaches an extremely significant difference(P < 0.01);The expression of GFAP in the drug-treated group is lower than it in the model group and the comparison gets a very significant difference(P?0.01).The expression in model group gets the highest among three groups.3 The index of the expression of synapse related protein: MAP-2expression: The expressions of MAP-2 in the drug group and sham operation group are higher than it in the model group and the comparison has significant differences(P?0.01,P?0.05).The expression of MAP-2 in the drug group is significantly higher than it in the sham operation group(P < 0.01);The expression of SYP: The expression of SYP in the drug group is significantly higher than it in the model group and the sham operation group,the comparisons get very significant differences(P < 0.01,P < 0.01);When the drug-treated group compared with the sham operation group,the expression of SYP also reaches a very significant difference(P < 0.01).The expression of SYP in the sham operation group is significantly higher than it in the model group(P < 0.01);The expression of PSD-95: The expressions of PSD-95 in the sham operation group and drug-treated group are significantly higher than it in the model group and the comparisons are very significantly different(P <0.01,P < 0.01),it gets a highest level in the drug-treated group,when compared with the sham operation group,the difference is statistically very significant(P < 0.01).Electron microscopic observation of the synapse: In the sham operation group,the synaptic gaps are clear and the outlines of Pre and post synaptic membrane are intact.There are abundant synaptic vesicles,the surface ofsynapse is regular,and the Pre and post synaptic membrane are in good agreement,a thicker post membrane can be seen.Mitochondria are intact,endoplasmic reticulum have more ribosomes.In the model group,the synaptic gaps are blurred,the anterior and posterior membranes are swelling and cavitating.It is difficult to distinguish between presynaptic and postsynaptic membrane.The number of synaptic vesicles decrease,mitochondrions are pyknosis and get less.Endoplasmic reticulums degranulate.In the drug-treated group,the structures of pre and post synaptic membrane are complete.The synaptic gaps are clearly visible,the synaptic vesicles are abundant,there are abundant and intact mitochondrions.Endoplasmic reticulums have plenty of particles.[Conclusion]: MDPs has obvious therapeutic effect on VD,the study shows that the mechanism lies in: 1.Through countering apoptosis of hippocampal neurons,inhibiting inflammation,promoting neuron regeneration to protect and restore the inherent construction model of neuro-vascular unit in the ischemic environment.2.Through inhibition of astrocytes proliferation and promoting oligodendrocytes proliferation and axon myelination to interfere with glial remodeling so as to ensure effective conduction of neural information;3.Through preventing synaptic damage and degeneration in cases of ischemia,promoting synaptic reconstruction to ensure synaptic plasticity and neural information accurate delivery. |
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