Experimental Study On The Treatment Of Vascular Dementia With Cells Transplantation And Medicine

PART IExperimental Study on the Treatment of Vascular Dementia withCholinesterase InhibitorObjective: To investigate the effect of cholinesterase inhibitor on cognitive function of chronic cerebral hypoperfusion rats, and discuss its cholinergic mechanism.Methods: The values of cholinesterase lever and density of cholinergic fibres together with the results of experiments in water maze and the values of event related potentials (ERP) were recorded in four groups as following: normal group, chronic cerebral hypoperfusion group (vascular dementia group performed by bilateral common carotid artery ligation) , control group (physiological saline, 3.5ml/(Kg·day) for six weeks) and Aricept treatment group (0.03%Aricept, 3mg/(kg·day) for six weeks).Results: The result of water maze experiment and the value of P300 latency of chronic cerebral hypoperfusion group are worse than those of normal group (P<0.05). The decrease in cholinesterase lever and density of cholinergic fibres in cortex of frontal lobe and hippocampus zone is observed in chronic cerebral hypoperfusion group, which is more significant than that in normal group (P<0.05). The result of water maze experiment and the value of P300 latency of Aricept treatment group are improved compared with the chronic cerebral hypoperfusion group and the control group (P<0.05), but cholinesterase lever and density of cholinergic fibres in cortex of frontal lobe and hippocampus zone decreased severely than chronic cerebral hypoperfusion group and the control group (P<0.05). Conclusion: Chronic cerebral hypoperfusion can influence the cognitive function significantly. The decline of cognitive function is coincident with the decline of concentration of cholinesterase and density of cholinergic fibres. It is well known that the measurement of P300 latency is an objective method which can be used to observe the change of cognitive function and the effect of medicine treating dementia. The P300 latency measurement also confirms the impairment of cognitive function of chronic cerebral hypoperfusion rats by detecting. Both ethology (maze experiment) and electrophysiology (P300 latency) confirm that Aricept has positive effect on prevention and curing the injury of cognitive function produced by chronic cerebral hypoperfusion. The protective effect is achieved by inhibiting cholinesterase, thereby increasing acetylcholine. It is a doubt that the protective effect is associated with the increase of cerebral blood flow and activation of cholineacetyl transferase which are all induced by the Aricept. Part II Make Aging Vascular Dementia Rat ModelObjective: To make aging vascular dementia rat model which pathology is approaching to the pathology of human vascular dementia.Mothods: Model group rats were performed unilateral carotid ligation twice in 72hours after they had been injected D-galactose for four weeks. Infarction areas and breakdown of blood brain barrier of model group were seeked. The following four parameters of normal group and model group will be observed after 6 weeks: 1, Morris water maze; 2, P300 latency; 3, numbers of pyramidal cells in CA1 zone; 4, the density of synaptophysin in CA3 zone.Results: Comparing to the traditional bilateral carotid ligation, the improved operation has less mortality (17%) . Results of ethology (Morris water maze experiments, P<0. 05) and electrophysiology (P300 latency, P<0. 05) proved an obvious impairment of cognitive function in model group. Both the amount of pyramidal cells in CA1 zone and the density of synaptophysin in CA3 zone decreased observably in model group (P<0. 05). TTC stain results showed that apparent infarction had not been found in model group rats. Evans blue stain result showed that blood brain barrier was not been broken 24 hours after bilateral carotid ligation in model group rats.Conclusion: The improved operation has less mortality, and further more, simulates successfully the characteristics of aging vascular dementia, which pathology is close to the pathology of human vascular dementia. This new model provides a better stage to research the etiopathogenesis and the treatment of vascular dementia. Blood brain barrier was not been broken 24 hours after bilateral carotid ligation in model group rats. Part IIIEffects of Allogeneic Bone Marrow Stromal Cells Transplantation on Aging Vascular DementiaObjective: To study the cognitive function improvement and histomorphology change in brain of aging vascular dementia rat which have been transplanted bone marrow stromal cells. This experiment results will provide new proofs to exploring a kind of new treatment method which is using bone marrow stromal cells transplantation on vascular dementia.Methods: Bone marrow stromal cells were cultured and suspended in PBS at a concentration of 10×10~6 cells/μl. By using bregma as a landmark, ≈5.0×10~6 cells were slowly injected into each subventricular zone of aging dementia rats by using a stereotactic device. We repeated the intraperitoneal injections by using BrdU every day in 6 weeks. At 6 weeks posttransplant, rats were killed. We compared the results of Morris water maze, P300 latency, cell proliferation (using BrdU immunohistochemistry stain), numbers of pyramidal cells (using hematoxylin and eosin stain) in CA1 zone and the density of synapse (using synaptophysin immunohistochemistry stain) in CA3 zone of all four group rats.Results: The results of Morris water maze and P300 latency suggested the improvement of cognitive function in treatment group mice (P<0.05). It proved a positive effect of bone marrow stromal cells transplantation on aging vascular dementia in two directions including ethology and electrophysiology. The cell proliferation in subventricular zone was more obvious in treatment group rats than that in control group rats. In treatment group rats, the amount of pyramidal cells in CA1 zone and the density of synapse in CA3 zone were more than that in control group rats.Conclusion: It is well known that there is a protective effect of bone marrow stromal cells transplantation on cerebral infarct model, however, it is rarely reported protective effect on aging vascular dementia model. The cognitive function of aging vascular dementia rat which have been transplanted bone marrow stromal cell have been improved. The function remolding of brain may contribute great to cognitive function improvement. The remolding includes changes of numbers of pyramidal cell and density of synapse and it needs further research. Part IVStudy of the Protective Reason of Treating Aging Vascular Dementia Rat with Bone Marrow Stromal CellObjective: To study the possible mechanisms of cognitive function improvement on the aging vascular dementia mice which have been transplanted bone marrow stromal cell.Methods: Bone marrow stromal cells were cultured and stained with hochest33258. The blue fluorescent cells were suspended in PBS at a concentration of 10×10~6 cells/μl. By using bregma as a landmark, ≈5.0×10~6 cells were slowly injected into each subventricular zone of aging dementia mice by using a stereotactic device. At 6 weeks posttransplant, mice were killed. We observed the distribution and the differentiation of bone marrow stromal cells in rats brain (using fluorescence immunohistochemistry stain), and detected the expression of NGF-mRNA (using RT-PCR) in subventricular zone.Results: Blue fluorescent bone marrow stromal cells were found surrounding lateral ventricular and diffusing a distance from that, in the meanwhile, it differentiated into neuron-like cells and astrocyte-like cells in frontal lobe cortex and subventricular zone. The expression of NGF-mRNA increased after the rats had been transplanted bone marrow stromal cells.Conclusion: Transplantated bone marrow stromal cells correlate with host brain. It promotes cerebral function remolding of aging vascular dementia mice by means of secreting NGF and promoting differentiation of itself and neural stem cells.