| Objective: As an acquired syndrome of intelligent impairment, vascular dementia(VD), which is a kind of cerebral dysfunction caused by various kinds cerebral vascular disease, demonstrates mainly as learning and memory dysfunction, accompanied with the possible disorder of tongue, motion, direction and personality. With the increase of the proportion of the elderly in the population and that of the curing rate of cerebral vascular disease, morbidity of VD mounts continuously and hence brings about heavy loads to the society and family. However, both the pathogenesis of VD and its specific treatment remain unknown up to now. It is therefore significant to study the pathophysiological mechanism of VD and to find some effective treatments .Evidence emerging within the last decade has shown that glutamate excitotoxicity is presumed to be involved in pathological mechanisims in vascular dementia. It is very important to attenuate the injury of excitatory amino acid as well as ischemic damage and learning and memory. The excitator aminoacid transporters (EAATs) provide the primary mechanism for the reuptake of glutamate from the extra-cellular space, which will help to prevent over-activation and cytotoxicity. Hypoxia and/or ischemia can induce the release of glutamate. Such increased levels of glutamate, and the subsequent activation of ionotropic NMDA receptors, are primarily responsible for an increase in intracellular Ca2+, Ca2+ overload intracellular can active Calpain, which induce overactivation of enzyme and dysfunction of cells. Recent advances suggest roles for cyclin dependent kinase 5(Cdk5) in vesicle cycling, ion channel modulation and intracellular signalling. These data are correlated with others implicating Cdk5 in synaptic plasticity, learning and memory. p35, an activator of Cdk5, is also a substrate of Calpain. Under pathological condition, p35 can be cleavaged by Calpain to p25 protein, the latter causes hyperactivation of Cdk5, leading to neurodegeneration via several cell death paradigms including apoptosis, excitotoxicity and neurifila- ment hyperphosphorylation. Oxidative damage caused by reactive oxygen species(ROS) has been proposed to be critically involved in cognitive dysfun- ction. The oxidant-antioxidant status of neurons was assessed by determine- ing the levels of superoxide dismutase(SOD) activity and malondialdehyde (MDA). Oxidative stress and other perturbation to neurons can result in the calpain-mediated cleavage of p35 to p25. On the other hand, deregulated Cdk5 may cause oxidative stress by compromising the cellular anti-oxidant defense system. Recent evidence indicates that under ischemic and hypoxic episodes, the oxidative stress induced by free radicals take part in the dysfun- ction of mitochondria and cell death. To date much less is known about the role of EAATs, Calpain-Cdk5 and ROS signal transduction cascades in VD.Donepezil hydrochloride has been used widely in the treatment of VD, but the pharmacological mechanism of treating VD is still in discussion. Donepezil can inhibit acetylcholinesterase(AchE) to degrade to acetylcholine (Ach) reversibility, increase quantity of Ach, which affect neuronal conduction and increase the ability of learning and memory . Extensive research revealed that donepezil can increase cerebral blood flow, activates the express of ERK, and decrease express of subtype of NMDAR1-NR1, increase NR2B express of hippocampus, affect expression of MAPK mRNA to adjust learning and memory. These results indicates that donepezil can increase learning and memory function by multiple pathway besides inhibiting AchE. To date the role of donepezil on EAATs, Calpain-Cdk5 pathway and oxidative stress in VD have not been studied in detail. The present study aims to examine the involvement of the EAATs, Calpain-Cdk5 pathway and oxidative stress in VD mice, in order to elucidate the potential effect of donepezil in treating VD.Method:(1) Kunming mice were subjected for continuously three repeated times ischemia-reperfusion through the ligation of the bilateral common carotid arteries, accompanied by sham group and drug group (donepezil hydrochloride). The capability of learning and memory of mice were investigated by the stepdown test and water maze test, the pathological change of hippocampus in each group were observed by HE staining. (2) The tissue were dyed by immunohistochemistry technique to observe the expression of EAAT1 and EAAT2. (3)The expression of CalpainⅠin hippocampus were studied by immunohistochemistry. (4) The expression of Cdk5mRNA and protein were studies by RT-PCR and Western-blot technique. Western-blot was used to observe the expression of p25 protein. (5) Ultraviolet method was used to evaluate the activity of SOD and the content of MDA.Results:1 Behavioral test and pathology change of VD mice1.1 Learning ability of step-down test:The response time of model group 29d, 43d and 57d postsurgery were (120.46±30.25),(133.16±29.77)and(121.05±31.43) respectively, longer than sham-operated group(29.38±3.76), (28.33±4.28)and (28.72±2.67) (P<0.05); The response time of donepezil-treated group were(48.12±11.23), (56.12±12.44)and(42.17±11.26) respectively, shorter than model group (P <0.05). The number of error of model group 29d,43d and 57d were (3.88±1.29), (4.38±1.13)and(3.14±1.29) respectively, with that of sham- operated group were(1.96±0.46),(2.16±0.43) and (1.58±0.63). The number of error of model group were much more than sham-operated group (P<0.05); The number of error of donepezil-treated group were(2.27±0.64), (2.86±0.87) and (1.96±0.78)respectively, lower than model group(P <0.05).1.2 Memory ability of step-down test:The latent time of model group 30d,44d and 58d postsurgery were(79.97±26.38), (80.78±26.12) and (79.87±20.17) respectively, shorter than that of sham-operated group (195.78±36.55), (186.78±36.21) and (178.26±35.23)(P<0.05). The latent time of donepezil-treated group were (156.13±31.25), (161.22±33.15) and (129.58±22.43) respectively, longer than model group(P <0.05). The number of error of model group at 30d, 44d and 58d were(4.14±1.25), (4.04±0.98) and (3.98±1.14)respectively, more than sham-operated group (1.93±1.01), (1.91±0.56) and (1.76±1.06) (P<0.05). The number of error of donepezil-treated group were (2.56±1.08), (2.74±0.87) and (1.89±0.86) respectively, lower than that of model group (P <0.05).1.3 Learning ability of Morris water maze test:The swimming time of model group 29d,43d and 57d post surgery were(124.33±53.22), (116.21±50.31) and (119.31±55.41)respectively, longer than that of sham-operated group (66.32±51.61), (61.63±38.86) and (65.73±51.96) (P<0.05). The swimming time of donepezil-treated group were(80.56±53.28), (76.43±43.86) and (77.49±48.86) respectively, shorter model group(P <0.05). The number of error of model group 29 d,43d and 57d post surgery were(11.68±1.36), (15.76±1.68) and (13.78±1.77) respectively, more than that of sham-operated group (5.76±1.55), (5.65±1.28) and (5.85±1.39)(P<0.05). The number of error of donepezil- treated group were(7.48±1.67), (7.38±1.34) and (7.38±1.49) respectively, lower than model group(P <0.05). There was no significant difference between sham-operated group and donepezil-treated group(P>0.05).1.4 Memory ability of Morris water maze test:The swimming time of model group 30d,44d and 58d post surgery were(111.58±48.32), (109.77±51.28) and (111.32±58.38) respectively, longer than sham-operated group (51.23±45.75), ,(49.34±36.36) and (49.56±40.33) (P<0.05). The swimming time of donepezil-treated group were (76.29±41.27), (66.27±40.86) and (57.27±40.11) respectively, shorter than that of model group(<0.05). The number of error of model group 30d,44d and 58d post surgery were(11.69±2.34), (14.65±1.39) and (14.95±2.19) respectively, more than that of sham-operated group(5.31±1.22), (5.35±1.04) and (5.68±1.13)(P<0.05). The number of error of donepezil- treated group were(6.33±1.46), (7.01±1.26) and (7.07±1.35) respectively, lower than model group(<0.05). There was no significant difference between sham-operated group and donepezil-treated group (P>0.05).1.5 HE staining of hippocampus: (1) Sham-operated group:The hippocampal profile was clear, there were 3 to 5 layers pyramidal cells, circinal and big nucleus, clear nucleolus. The many pyramidal cells ranged tightly, and dense nerve fibers were observed. The number of normal neuron 4, 6 and 8 weeks post surgery were (101.58±15.32), (108.24±11.33) and (101.24±12.26).(2) Model group:There were decreased layers pyramidal cells that ranged loosely. Their nucleus that had not clear structure became small, were dyed strongly, nerve fibers ranged unregularly. The number of normal neuron 4, 6 and 8 weeks post surgery were(65.33±19.28), (21.23±3.12) and (50.52±11.28), lower than sham-operated group(P<0.05), among those the most damaged group were 6 weeks post surgery, there are only e few neuron alive, the number of normal cell were lower than that of 4 and 8 weeks post surgery (P<0.05).(3) Donepezil-treated group:Above mentioned pathologic changes eased off in drug group. The pyramidal cells ranged tightly at 4 and 8 weeks post surgery. The number of normal neuron were(88.22±19.88) and (83.36±12.21), higher than that of model group(P<0.05). There was no significant difference between sham-operated group and donepezil-treated group(P>0.05). At 6 weeks post surgery, the pyramidal cells ranged tightly, but still with some nucleus shrinkage. The number of normal neuron were(46.38±5.17), higher than that of model group (P<0.05), but still lower than sham-operated group (P<0.05). These results suggest that hippocampus is an important region relating to learning and memory. Cerebral ischemia and reperfusion can induce the occurrence of VD, with hippocampus neurons damaged severely, among which the worst is emerging at 6 weeks post surgery. Donepezil can reduce the pathological changes and the ability of learning and memory of VD.2 Expression of EAATs in hippocampus of VD mice and the effect of donepezilThe mice's cerebral tissue were fast taken out, fixed up via perfusion of 4% paraformaldehyde solution. Then paraffin–embedded, coronal and serial sections were taken from each brain and EAAT1 and EAAT2 were stained through immunohistochemistry. The result revealed that:(1) In model group, the expression of EAAT1 on membrane of cells 4 weeks, 6 weeks and 8 weeks post surgery were (0.099±0.009), (0.122±0.009)and(0.112±0.010), higher than sham-operated group(0.030±0.003), (0.031±0.002)and(0.030±0.003)(P<0.05), with the highest expression was at 6 weeks post surgery; the expression of EAAT1 in donepezil-treated group at the three time points were (0.047±0.004), (0.046±0.003)and(0.044±0.004), lower than model group (P<0.05), there was no significant difference between sham-operated group and donepezil-treated group (P>0.05). (2) In model group, the expression of EAAT2 at 4 weeks,6 weeks and 8 weeks post surgery were (0.098±0.008),(0.113±0.008) and (0.101±0.009), higher than sham-operated group (0.031±0.003), (0.030±0.002)and(0.030±0.003) (P<0.05), the expression of EAAT2 in donepezil-treated group at the three time points were (0.039±0.003), (0.047±0.005) and (0.042±0.004), lower than model group(P<0.05), there was no significant difference between sham-operated group and donepezil-treated group(P>0.05). These results suggest that EAAT1 and EAAT2 were involved in the occurrence of VD, donepezil may improve learning and memory ability through reducing the expression of EAAT1 and EAAT2.3 Expression of Calpain I in hippocampus of VD mice and the effect of donepezilThe mice's cerebral tissue were fast taken out, fixed up via perfusion of 4% paraformaldehyde solution, then paraffin–embedded, coronal and serial sections were taken from each brain and Calpain I was stained through immunohistochemistry. The result revealed that: in model group, the expression of Calpain I in plasm of neurons 4 weeks, 6 weeks and 8 weeks post surgery were respectively(0.099±0.009, 0.121±0.010 and 0.115±0.010), higher than sham-operated group(0.030±0.003, 0.031±0.003 and 0.029±0.003) (P<0.05), with the highest expression at 6 weeks post surgery. The expression of Calpain I in donepezil-treated group at the three time points were (0.040±0.004, 0.044±0.004and0.042±0.003), lower than model group (P<0.05), there was no significant difference between sham-operated group and donepezil-treated group(P>0.05). These results suggest that cerebral ischemia can induce Ca2+ overload intracellular, with sustain high expression of Calpain I, paralleled with hippocampus neuronal damage. Donepezil may protect neurons from injury by reducing the expression of Calpain I.4 The Cdk5 mRNA and protein expression in hippocamous of VD mice and the effect of donepezil4.1 Protein expression of Cdk5 in hippocamous by Western-blotThe protein expression of Cdk5 in model group 4, 6 and 8 weeks post surgery were (0.54±0.05), (0.73±0.07) and (0.70±0.06), higher than sham- operated group (0.23±0.02), (0.31±0.02) and (0.33±0.02) (P<0.05), suggesting that the protein expression of Cdk5 was increased within 8 weeks after cerebral ischemia and reperfusion. In donepezil-treated group, the protein expression of Cdk5 were (0.28±0.02), (0.33±0.03) and (0.38±0.02), lower than that of model group and there was significant difference(P<0.05).4.2 Cdk5 mRNA expression in hippocamous by RT-PCRThe Cdk5 mRNA expression in model group 4, 6 and 8 weeks post surgery were (0.80±0.07), (0.91±0.08)and(0.92±0.08), higher than sham- operated group (0.35±0.02), (0.38±0.04) and (0.36±0.03) (P<0.05), suggesting that Cdk5 mRNA expression was increased within 8 weeks after cerebral ischemia and reperfusion. In donepezil-treated group, the mRNA expression of Cdk5 were (0.33±0.02), (0.40±0.04)and(0.39±0.08), lower than that of model group and there was significant difference (P<0.05).The result mentioned above indicate that the mRNA and protein expression of Cdk5 were increased from 4 weeks to 8 weeks after cerebral ischemia and reperfusion, this is accordance with behavioral test and neuronal damage, and with the expression of Calpain I, which is upstream of Cdk5. It is reasonable to consider that abnormal increase expression of Calpain I-Cdk5 pathway take part in the hippocampus neuronal death of VD mice after cerebral ischemia, and maybe correlate to memory injury. Donepezil may improve learning and memory ability by reduce the expression of Calpain I and Cdk5.5 p25 expression of VD mice and the effect of donepezilThe expression of p25 in model group 4, 6 and 8 weeks post surgery were (0.44±0.04), (0.51±0.04)and(0.55±0.06), higher than that of sham- operated group (0.19±0.02), (0.24±0.02) and (0.20±0.02) (P<0.05), suggesting that the protein expression of p25 was increase within 8 weeks after cerebral ischemia and reperfusion. In donepezil-treated group, the p25 expression of were (0.26±0.02), (0.25±0.03) and (0.21±0.02), lower than model group and there was significant difference(P<0.05). As an abnormal activator of Cdk5, p25 high expression indicating irregulation of Cdk5, which is one of the most important case in evoking cell death, donepezil can reduce the expression of Calpain I and the Calpain-mediated generation of p25.6 The changes of SOD activity and MDA content in VD mice and the effect of donepezil6.1 The activity of SODSOD activity in model group 4, 6 and 8 weeks post surgery were (7.17±1.66 U/mg*prot), (7.07±1.43 U/mg*prot) and (7.23±1.33 U/mg*prot), higher than sham-operated group (3.10±0.86 U/mg*prot, 3.22±0.92 U/mg* Prot and 3.19±0.90 U/mg*prot) (P<0.05). In drug group, the SOD activity were (4.39±0.98 U/mg*prot), (4.88±0.97 U/mg*prot) and (4.76±0.95 U/mg* prot), there was no significant difference between the two groups(P<0.05). 6.2 The content of MDAMDA content in model group were(15.26±3.23, 16.33±3.76 and 16.67±3.75 nmol/mg*prot), higher than sham-operated group (6.33±1.16, 6.87±1.06 and 6.63±1.11 nmol/mg*prot) (P<0.05). In drugl- treated group, MDA content were (7.42±1.89, 8.32±1.68 and 8.58±1.76 nmol/mg*prot), lower than model group and there was significant difference (P<0.05).Both SOD activity and MDA content were increase in model groups, suggesting that oxidative stress is a key feature of the neurodegenerative process of VD. Donepezil , as a potent free radical scavenger, might protect neurons against oxidative stress by regulating the activity and expression of antioxidant enzymes.Conclusion:(1) This study has successfuly established VD model. The pathological features in hippocampus show that pyramidal cells are damaged, especially at 6 weeks post surgery. This model might mimic the cognitive deficiency of VD in clinic. Donepezil hydrochloride might improve learning and memory ability of mice by attenuate the injury of neurons.(2) It was confirmed by immunohistochemistry that sustain increased expression of EAAT1 and EAAT2 during 8 weeks post surgery in hippocampus were related to cognitive damage and neuronal damage in VD. This indicate that glutamate excitotoxicity is a main factor in pathogenesis of VD.(3) As an importmant content of Ca2+ -signal cascade, Calpain I expression is sustain high during 8 weeks post ischemia-reperfusion, accompanied by neuronal injury. Cdk5 expression is also increased demonstrated by immunohistochemistry method,RT-PCR and Western-blot technique, with high expression of its activator p29 protein, indicating that more p35 is cleavaged to p25 by Calpain, which induce dysregulation of Cdk5 and neuronal death. These results suggest an importmant role of Calpain I-Cdk5/p25 pathway in the occurrence of VD.(4)Ultraviolet results demonstrate that SOD and MDA might be involved in the occurrence of VD, indicating that oxidative stress play an important role in dementia after cerebral ischemia.(5) The cognitive abilities of mouse with VD could be improved by Donepezil hydrochloride through reducing the expression of Calpain I-Cdk5/p25 pathway, EAAT1 and EAAT2 and the production of ROS, this result suggest that Donepezil hydrochloride might have neuroprotective effects.
|
PDF Full Text Request