The Research Of Cholinergic Deficiency Mechanism In The Experimental Vascular Cognitive Impairment

Background and objects:Studies have shown that many central cholinergic mechanisms involved in the pathogenesis of VaD. Choline acetyltransferase (CHAT), acetylcholinesterase (ACHE), vesicular acetylcholine transporter (VACHT) is an important part of the cholinergic system, maintains acetylcholine (ACH) homeostasis. CHAT is especially synthase Ach, so central cholinergic neurons marker enzyme. This article will explore spatial learning and memory ability of VaD rats,CHAT, ACHE, VACHT protein expression in hippocampus, and observe the effect of donepezil, DL-3-n-butylphthalide (NBP), Hydroxy safflower yellow A (HSYA), fingolimod (FTY720) on spatial learning and memory ability of VaD and BDNF, VEGF, CHAT, ACHE,VACHT protein expressin in hippocampus, thus providing a new target and the theoretical basis for drug treatment of VaD.Methods:In this study, bilateral common carotid artery ligation (2VO) rat model of VaD, using Morris water maze was used to detect spatial learning and memory in rats VaD, for the first time while applying western blot method, immunofluorescence staining to detect CHAT, ACHE,VACHT protein expression in hippocampus of VaD rats, and to observe the relationship between cholinergic mechanisms and VaD. And apply donepezil,NBP, HYSA, FTY720on VaD rats while applying western blot method, immunohistochemistry or immunofluorescence staining method to detect effects of these drugs on rat hippocampal CHAT, ACHE, VACHT, VEGF, BDNF expression, and to observe effects of these durgs on spatial learning and memory abilities and cholinergic system of VaD rats.Results:1:1.1When compare to the normal group and sham group,model rats daily escape latency was significantly longer (P<0.05). Percentage target quadrant of model group rats was significantly lower than the normal group and sham group (P <0.05).1.2CHAT, ACHE, VACHT protein expression in model rats hippocampus was obviously downregulated.1.3HE staining:Hippocampus of model group, the number of cell layers of the hippocampus and reduce disorganized, cell volume was significantly reduced, some cells showed vacuolar degeneration, a large number of cell death, visible karyopycnosis nuclear rupture, cell protrusion structure is not clear. or even disappear. There are significant differences compared with the normal group.1.4Immunofluorescence staining:CHAT, ACHE, VACHT expression in model group hippocampus was significantly reduced.2:2.1Navigation test showed that rats of each group were trained by five days to establish a spatial memory. Space exploration experiments showed that donepezil group was significantly higher than the percentage of the target quadrant model group (P<0.05). The target quadrant percentage of NBP group was significantly higher than the model group (P=0.018). The percentage of the target quadrant of HYSA group was significantly higher than model group, but the difference was not statistically significant (P=0.053). The percentage of the target quadrant of FTY720group was higher than that of model group, but the difference was not statistically significant (P=0.018).2.2Western blot results showed that:In addition to donepezil treatment groups hippocampal CHAT, ACHE, VACHT expression were increased compared with the model group. ACHE expression in the treatment group compared with the donepezil group of mild reduction, CHAT, VACHT still higher than that in the model group. Compare with the model group group, BDNF expression in FTY720group, NBP group increased. VaD model group VEGF expression compared with the sham group was significantly increased. Donepezil group reduce the expression of VEGF is the most obvious.2.3HE staining:Each intervention group and the number of cell layers of rat hippocampus were normal, clear arrangement, no cell volume was significantly reduced, no significant vacuolar degeneration, only a small number of cells can be seen karyopycnosis nuclear rupture, cell projections are arranged relative neat.2.4Immunohistochemical staining:2.4.1VEGF immunohistochemistry-positive cells were seen in hippocampal CA1and CA3regions of the rats. VaD model group compared with the sham group was significantly increased.Donepezil group compared with model group to reduce the most obvious expression of VEGF. VEGF slightly decreased in HYSA group,FTY720group,when compare with model group.2.4.2BDNF immunohistochemistry-positive cells were mainly concentrated in the hippocampal CA1, CA3pyramidal cells and DG granule cells. Compared with the control group and the sham group, model group, BDNF expression slightly decreased immune response. NBP group, FTY720groups compared with model group, the positive expression of BDNF immunoreactivity was significantly increased.Conclusions:1. Bilateral carotid artery ligation (2VO) successfully established rat VaD model which can effectively simulate the human brain hypoperfusion resulting in diffuse pathological changes caused by ischemia and hypoxia, with the stability repeatability.2., CHAT, ACHE, VACHT protein expression in VaD rat hippocampus decreased, down to its consistent performance in learning and memory, suggesting CHAT, ACHE, VACHT reduced expression may be involved in the pathogenesis of VaD3. Donepezil can significantly improve the spatial learning and memory VaD in rats, increased expression of hippocampal CHAT、VACHT,reduce the expression of ACHE, while reducing the expression of VEGF.Cholinesterase inhibitors,Donepezil may not only increase directly acetylcholine content but also inhibit the inflammation and protect cholinergic neurons.4. NBP can significantly improve VaD rats’ spatial learning and memory ability and increase their CHAT, ACHE, VACHT,BDNF expression in hippocampus. NBP may be able to protect the cholinergic neurons and promote the function of cholinergic system by BDNF.5. HSYA can improve rats’spatial learning and memory ability and increase the expression of CHAT, ACHE, VACHT in rats" hippocampus, slightly decrease the expression of VEGF. HSYA may protect hippocampal cholinergic neurons not only by vascular mechanisms, but also by reducing inflammatory damage.6. FTY720can improve spatial learning and memory ability of VaD rats, also increase the expression of BDNF CHAT, ACHE, VACHT in rat hippocampus. FTY720is immunomodulator which can reduce ischemic disease early inflammatory injury and increased expression of BDNF, thereby protecting the cholinergic neurons, thus improving the cognitive function of VaD rats.