Relationship Between Hippocampal NMDA Receptor Together With Associated Synaptic Proteins And Cognitive Impairment Of Vascular Dementia Rats

Vascular dementia(VD) is a syndrome secondary to the cerebral damage caused by a series of cerebrovascular events, which displays the impairment of learning and memory. Being one of the main types of senile dementia, VD is commonly found in the old people. And with the aging of the society and the changing of food variety, the incidence of VD is rising, which has attracted more and more researchers to focus on this topic. Recently, chronic cerebral hyoperfusion is regarded as the main cause of VD. So, in this experiment, we establish VD rat model by permanent, bilateral occlusion of the common carotid arteries(2-VO) to study the relationship between hippocampal NMDA receptor together with associated synaptic proteins—post synaptic density-95(PSD-95) and calcium/calmodulin-dependent protein kinase II(CaMK II)and cognitive impairment of vascular dementia rats. Meanwhile, we also investigate the role of memantine, a NMDAR antagonist, in VD therapy in order to find more ways to prevent and cure VD.1. Grouping of experimental animals and establishment of animal modelWistar rats weighing 280-350g(3～5months old) were randomly divided into sham-operated group, VD model group, memantine-treated group. According to the feeding time after operation, the sham-operated group and VD model group were individually assigned into 8 subgroups, i.e. 1d, 3d, 1w, 2w, 4w, 8w, 12w and 16w after operation. Corresponding with the subgroups of 4w, 8w, 12w and 16w after operation, in the treated groups, memantine was administered intragastrically at a high amount of 10 mg/kg/d and a low amount of 5 mg/kg/d respectively in 4 weeks prior to sudden death.VD rat model was set up by disconnecting bilateral common carotid arteries and ligating distal and proximal end of the bilateral common carotid arteries,then cutting them from the middle. However, in the sham-operated group, we only disconnected bilateral common carotid arteries without ligating and cutting them.2.Investigations of ethology and histology of VD rat modelThe abilities of learning and memory of all the rats were determined by Morris Water Maze(MWM) prior to operation. The shorter the escape latency and the more the spanning platform times they had, the better the ability of learning and memory they possessed. After operation, as the ischemic time prolonged, the escape latency of VD model rats became longer and the spanning platform times became less. The MWM results of VD model groups of 4w, 8w, 12w, 16w after operation were greatly different from that of the sham-operated group(P<0.01), which suggested that VD model rats induced by 2-VO had became demented since 4w after operation and continued until 16w after operation. Histologically, HE staining of the sham-operated group showed neurons of frontal lobe cortex and pyramidal cells of hippocampus lined up in order and uniformity,whose cellular structures were integrity. However, HE staining of VD model groups showed at the beginning,the diffused cerebral edema and exudation were apparent,then attenuated gradually. Subsequently the degeneration and detachment of neurons could be seen,so did the necroses of the neurons and hypertrophic neuroglial cells and hyperplasy of small vessels. Accordingly, we confirmed that 2-VO could lead to the chronic, persistent, ischemic injury similar to VD, which was parallel to the impairment of intelligence.Among the treated group, there was no difference in the MWM results and histologic characters between the sham-operated group and the subgroup of 4w after operation in which memantine was administered at the same time as operation, irrespective of the amount of memantine. In other 3 subgroups, only the rats with high dosage memantine had signicantly improved MWM results and histologic characters, compared to VD model group(P<0.01 or 0.05). But when compared with the sham-operated group, these 3 subgroups didn't recover to normal state (P<0.01). Therefore, we concluded that memantine could protect neurons from the ischemic and anoxic damage to some degree and its effect depended closely on the first time to employ memantine and its dosage.3. The mechanism of alterations of NR2B subunit expression of vascular dementia ratsThe change of the expression of NR2B in hippocampus was investigated by immunohistochemical staining, Western blot and RT-PCR. With the ischemic time prolonging, the expression of NR2B presented a curve that firstly rised and then dropped. In detail, the expression grew significantly 3d after operation and reached its peak at 1-2 weeks after operation(P<0.01), and fell down gradually, then reduced to the minimum at 16 weeks after operation(P<0.01). Combining with the MWM results which showed VD model rats became demented at 4w after operation, we thought that during the early period of ischemia, the impairment was concerned with neurotoxicity derived from the excessive expression of NR2B, however; during the advanced stage, the insufficient NR2B which was unable to perform its physiological function led to the defect of learning and memory.As a noncompetitive NMDAR antagonist with low affinity, memantine was used to investigate its effect on the expression of NR2B. We found that at 4 weeks after operation, the rats with high dose as well as low dose exhibited no statistically difference in the expression of NR2B in contrast with the sham- operated group. However, at 8,12,16 weeks after operation, in the treated group with high dose, the expression of NR2B was better than that of VD model rats and worse than that of the sham-operated group(P<0.01 or 0.05). On the other hand, in the treated group with low dose, the expression was no better than that of VD model rats and far worse than that of the sham-operated group (P<0.01). These results suggested that during the early period of ischemia, memantine prevented neurotoxicity by interrupting NMDAR, however; during the advanced stage, memantine improved the function of NMDAR by up-regulating NMDAR. And its effect was closely related to drug dosage and administration time. We also found that there was no difference regarding the expression of NR2B at 4w, 16w after operation between the treated group with high dose and low dose. But the significant difference could be found at 8w, 12w after operation between the treated group with high dose and low dose(P<0.01 or 0.05). According to these, we inferred that when it was administered during the early period of ischemia, the amount of memantine was not strictly demanded; when it was administered during the advanced stage, relatively high amount of memantine and long course of therapy were necessary.4. The mechanism of alterations of PSD-95 expression of vascular dementia ratsThe change of the expression of PSD-95 in hippocampus was investigated by immunohistochemical staining and Western blot. With the ischemic time prolonging, the expression of PSD-95 showed the same tendency as NR2B, which firstly rised and topped at 2w after operation, then dropped and less than that of the sham-operated group(P<0.01). Because PSD-95 performed the function to integrate the synaptic signals mediated by NMDAR, we could understand that during the early period of ischemia, the expression of PSD-95 increased to transmit the neurotoxic signal mediated by the excessive expression of NMDAR; during the advanced stage, the decreased PSD-95 expression which weakened the connection with NMDAR in the learning and memory path resulted in the development of VD. This feature also meant PSD-95 might be employed as a new powerful target of VD therapy. Moreover, in the memantine-treated group, the expression of PSD-95 presented the same tendency as NR2B again, which meant that memantine avoided the excessive expression of PSD-95 during the early period of ischemia and prevented the expression from reducing during the advanced stage. In a word, we could infer that under the stress of 2-VO, NMDAR and PSD-95 experienced the same change as a complex and induced the alterations of the downstream conduction network, which brought the cascade reactions of NMDA receptor complex(NRC) and contributed to the ischemic necroses of neurons and the impairment of learning and memory.5. The mechanism of alterations of CaMK II activity of vascular dementia ratsThe change of CaMK II activity (including Ca²⁺-dependent and Ca²⁺- independent activity) was measured by 32P incorporation. In the sham-operated group, CaMK II activity was on the basis of Ca²⁺-dependent activity, which was in accordance with the fact that autophosphoralated CaMK II activity (Ca²⁺-independent activity) was little physiologically. However, in VD model group, as the ischemic time extended, Ca²⁺-independent activity firstly rised and then dropped and Ca²⁺-dependent activity firstly dropped and then rised. They both reached their poles at 2w after operation and eventually were less than the activity of the sham-operated group. Total CaMK II activity was the sum of Ca²⁺-dependent and Ca²⁺-independent activity and firstly increased and then decreased which presented the tendency similar to NR2B and PSD-95. Considering the pathogenesis of VD that ischemia led to dementia and the MWM results, we evaluated that Ca²⁺-independent activity was necessary for learning and memory while Ca²⁺-dependent activity was indispensable for guarding against neuronal injury caused by overloading of Ca²⁺. So the balance of Ca²⁺-dependent activity and Ca²⁺-independent activity made up the entire function of CaMK II, and the breakdown of this balance led to the development of VD. On the other hand, the excessive boosting of CaMK II activity was harmful to learning and memory. In the treated group, CaMK II activity restored to normal state only when memantine was administered at the same time as operation. When memantine was administered at other time, there was no obvious difference of activity between the treated group and VD model group, which was different from the results that memantine could ameliorate the expression of NR2B and PSD-95 at 8w, 12w, 16w after operation. As for this, the author estimated that CaMK II activation amplified the cascade reactions of ischemic signal mediated by NMDAR, and the water fall effect brought by signal transduction was irreversible. What's more, the change of CaMK II activity might be related to some other NMDAR-independent biochemical events induced by excitotoxicity, which needed further research to confirm. However, these results indicated that we should choose more targets in more ways to co-treat VD.