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Isopropylidene shikimic acid on synaptic plasticity in rats with chronic hypoperfusion

Author: WangJing
Tutor: SunJianNing
School: Beijing University of Traditional Chinese Medicine
Course: Pharmacology
Keywords: cAMP response element binding protein Choline acetyltransferase Brain-derived neurotrophic factor Synaptophysin Synaptic plasticity Isopropylidene shikimic acid
CLC: R285.5
Type: PhD thesis
Year: 2008
Downloads: 270
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Abstract


Vascular dementia (VD), which is characterized by progressive intellectual decline induced by ischemia hypoxia or hemorrhage brain lesion, represents the second most common dementia accounting for about a quarter to a half of all cases of dementia in developed countries. Chronic cerebral hypoperfusion (CCH) is one of common pathophysiologic basis of VD and Alzheimer’s disease. CCH impacts the delivery of glucose and oxygen, leading to the handicap of energy metabolism in the brain synaptic pathway, followed by the decline of the whole brain function including memory decline. The structural basis of memory is synapse. The enhancement of synaptic function contributes to the formation of memory. The rats with memory decline showed the changes in synaptic structural parameters, such as the decrease of numerical density of synapses (Nv) and docked vesicles (Dv).With increasing number of the elderly in world populations, dementia, characterized by progressive descending of memory and cognition, has given rise to enormous socioeconomic burden. In the past few decades, a great deal of investigation has been conducted. However, the efficiency of currently available strategies for the prevention of VD remains poor. 3,4-oxo-isopropylidene-shikimic acid(ISA)was extracted from Illicium verum Hook. Fil, a traditional Chinese herb. Our preliminary work showed that ISA could suppress variously experimental thrombosis and platelet aggregation significantly, improve the morphology and decrease the apoptosis rate of cultured cortical and hippocampal neurons after hypoxia-reoxygenation injury, exert neuroprotective effects on rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion by an intraluminal thread, as evidenced by decreasing the neurological severity score, infarct volume, brain water content and brain edema. It was indicated that the anti-free radical effects of ISA might account for the protection against cerebral ischemia. Thus, ISA appeared to be a promising drug to ameliorate cognitive deficits induced by cerebral ischemia. But to our knowledge, the effects and the mechanism of ISA on CCH in rats remain to be elucidated. In the present study, we made use of the permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) model of CCH to evaluate the effects of ISA on synaptic plasticity in dementia rats. The possible mechanism underlying its effect was also explored. To address this issue, a variety of multidisciplinary approaches including Morris water maze, transmission electron microscopy, immunohistochemical staining, Western blotting, fluorescent quantitative real-time polymerase chain reaction were used.There are three stories in this thesis.PartⅠCreation of two kinds of VD rat models and the investigation on the pathological changes in neurons and synaptic ultrastructure in hippocampal CA1 subfield Objective: To explore the characteristics of the photochemical model and 2VO model, and to investigate the pathological changes of neurons and synaptic ultrastructure in hippocampal CA1 subfield.Methods: A focal lesion in the left parietal cortex was induced photochemically by using Rose Bengal as a photosensitive dye and cold light beam, then the rats were treated with ISA orally once a day for 30 days. The cognitive effects of ISA were assessed in rats using the Morris water maze test for spatial learning and memory. HE staining and Nissl staining were used to study the neuronal alteration in left parietal cortex. For the reproduction of CCH as it occurs in VD, permanent occlusion of the bilateral common carotid arteries of rats (2-vessel occlusion, 2VO) was introduced. 60 days after 2VO surgery, the rats underwent spatial cognition test in Morris water maze. Subsequently the brain sections were subjected to HE staining and transmission electron microscopy. Results:①Photochemical lesion induced spatial cognition deficits, which was proven by prolonged escape latency on the 2-4 training days(p<0.05, p<0.01)and the larger initial angle in probe test, compared to the sham-operated group(p<0.05).②Photochemical lesion could induce round infarction in the left parietal cortex of rats. The neurons in the infarction almost disappeared. No obvious hemorrhage and edema existed. The pathological changes were gradiently severe from relatively normal brain region to the infarction.③8 weeks after 2VO surgery, the model rats showed significant cognition deficits, as evidenced by longer swimming path length on the third training day(p<0.05), the larger initial angles on the second and third training day(p<0.05, p<0.01), compared to the sham-operated group rats. In the probe test, the model rats exhibited larger initial angles(p<0.05), compared to the sham-operated group rats. No difference in the quadrant percentage was observed between the two groups(p>0.05).④Macroscopic evaluation of rat brains demonstrated that 2VO induced pyramidal neuron damage in hippocampus CA1 subfield 65 days after operation. The rats showed severe neuronal loss and the gliocyte proliferation in the hippocampal CA1 region.⑤The synaptic ultrastructures of neuropil field within CA1 stratum radiatum of the sham-operated group rats were intact. The synapses of GrayⅠtype showed clear synaptic clefts, typical asymmetric interfaces, intact structures of the pre- and postsynaptic membranes, round clear vesicles and thick postsynaptic densities (PSD). The synaptic ultrastructures of 2VO group presented the blurry synaptic clefts, swollen, vacuolated pre- and postsynaptic membranes and less synaptic vesicles in the synaptic terminals.⑥The mortality rate in the model rats with photochemical lesion was 25%, while 10% in the model rats with CCH. Conclusion:①Two VD models induced by the photochemical lesion and 2VO respectively in rats were created. The changes in the cognition and neuronal pathology in the 2VO rats were observed.②2VO model is more suitable for the investigating in the further pharmacodynamics research due to its lower mortality rate and easier surgical process.PartⅡPharmacodynamics research of 3, 4-oxo-isopropylidene-shikimic acid in rats with vascular dementia induced by chronic cerebral hypoperfusionObjective: To probe the effects of ISA on spatial cognition, neuronal pathology and synaptic ultrastructure within hippocampal CA1 subfield in rats with CCH.Methods: We used 2VO to induce CCH in rats. Male Sprague Dawley rats were randomly divided into six groups including vehicle-treated sham-operated group, vehicle-treated operated group, ISA 100mg/kg-treated operated group, ISA 50mg/kg-treated operated group, ISA 25mg/kg-treated operated group and Hydergine 0.6mg/kg-treated operated group. Vehicle group rats’bilateral carotid arteries were occluded. The sham-operated rats received the same operation with the exception of ligation. Other rats were intragastricly administered relevant drugs once per day. Eight weeks after 2VO, Morris water maze was used to measure spatial learning and memory. Each rat received two trials everyday for 4 consecutive days. Length of swimming path to escape onto the hidden platform and initial angle were recorded. On the fifth day, each rat was subjected to a 20-second probe trial in which the platform was removed and the initial angle and typical swim-tracking path were recorded. Subsequently, the morphological changes in neurons were evaluated with HE staining in hippocampal CA1 subfield. Transmission electron microscopy was used to evaluate the following synaptic structural parameters in hippocampal CA1 subfield. Nv, surface density (Sv), DV and DV per unit length of active zone (L) were measured.Results:①The sham-operated rats showed smaller initial angles on the second (p<0.05) and third (p< 0.01) training days, and the shorter path length on the third training day (p<0.05), compared to 2VO group. In the probe test, the initial angle of sham-operated group was smaller than 2VO rats (p<0.05). The swimming strategy was tendency at the early stage and straight at the late stage in sham-operated rats, and marginal at the early stage and random pattern at the late stage in 2VO rats. Administration of ISA at the dose of 100mg/kg could significantly shorten the path length on the second training day (p<0.05), but didn’t change the path length on other training days, and initial angle in navigation trails and probe trail, compared to 2VO group (all p>0.05). Administration of ISA at the dose of 50mg/kg could significantly reduce initial angle in probe trail (p<0.05), but didn’t change the path length and initial angle in navigation trails, compared to 2VO group (all p>0.05). Administration of ISA at the dose of 25mg/kg could significantly reduce initial angle on the third training day in navigation trail(p<0.01)and probe trail (p<0.05), but couldn’t change the path length in navigation trail, compared to 2VO group (all p>0.05). When treated with dihydroergotoxine at the dose of 0.6mg/kg, the rats showed smaller initial angle on the third training day, compared to 2VO group (p<0.05). No difference in swimming path length on other training days, and initial angle in probe trail were observed, compared to 2VO group (all p>0.05). The main swimming strategies in all drug-treated groups were tendency at the early stage and straight at the late stage.②Microscopic evaluation of rat brains demonstrated that 2VO induced pyramidal neuron damage in hippocampus CA1 subfield 65 days after operation. 2VO rats showed severe neuronal loss and the gliocyte proliferation in the hippocampal CA1 region. The structures of pyramidal neurons in all drug-treated groups were relatively intact.③The synaptic ultrastructures of neuropil field within CA1 stratum radiatum of the sham-operated group rats were intact. The synapses of GrayⅠtype showed clear synaptic clefts, typically asymmetric interfaces, intact structures of the pre- and postsynaptic membranes, round clear vesicles and thick PSD. The synaptic ultrastructures of 2VO rats presented the blurry synaptic clefts, swollen and vacuolated pre- and postsynaptic membranes. The synaptic ultrastructures were relatively intact in all drug-treated groups. There were more synaptic vesicles in the presynaptic terminals in ISA and dihydroergotoxine-treated groups, compared to 2VO group. Compared to the sham-operated group, the 2VO rats showed significantly reduced Nv, Sv, DV, DV/L and synaptic curvature (all p<0.01), but the thickness of PSD didin’t change markedly (p>0.05). All the ISA-treated and dihydroergotoxine-treated rats showed markedly increased Nv, Sv, DV and DV/L (p<0.05, p<0.01), compared to 2VO group. Administration of ISA at the dose of 50mg/kg (p<0.01) and 100mg/kg (p<0.05) could significantly increase the synaptic curvature.Conclusions: Administration of ISA could improve the decline of learning and memory, and protect the neuronal morphology of the brain in rats with CCH. It improved the structural plasticity of CA1 synapses.PartⅢProbe into the potential molecular mechanism of 3, 4-oxo-isopropylidene-shikimic acid underlying its synaptic plasticity effects on vacular dementia induced by chronic cerebral hypoperfusionObjective: To probe effects of ISA on the expressions of brain derived neurotrophic factor (BDNF), its receptor tyrosine kinase B(TrkB), Choline acetyltransferase (ChAT), synaptophysin (SYP), phosphorylated cAMP response element binding protein (p-CREB) in the mRNA and protein levels within hippocampal CA1 region of CCH rats. Methods: Male Sprague Dawley rats were randomly divided into six groups including vehicle-treated sham-operated group, vehicle-treated operated group, ISA 100mg/kg-treated operated group, ISA 50mg/kg-treated operated group, ISA 25mg/kg-treated operated group and Hydergine 0.6mg/kg-treated operated group. After the behavioral test, rats were perfused intracardially. The paraffin sections were used for immunohistochemistry staining to probe the expression of p-CREB, BDNF, TrkB, ChAT and SYP in protein level within hippocampal CA1 subfield. The proteins were extracted and used for evaluation of the semi-quantitative expression of p-CREB, BDNF, TrkB, ChAT and SYP in protein level. Fluorescent quantitative real-time polymerase chain reaction was employed to measure the mRNA expression level of BDNF, TrkB, ChAT, SYP and CREB.Results:①At week 9 after 2VO surgery, the immunoreactive products of p-CREB, some brown granules, distributed in the neucli of pyramidal neurons within hippocampal CA1 region. The expressions of p-CREB in protein level and CREB in mRNA level were higher in 2VO group than in sham-operated group (both p <0.05). Administration of ISA at the dose of 100mg/kg increased the CREB expression in mRNA level, compared to the 2VO group (p<0.05). Dihydroergotoxine and ISA at three doses didn’t significantly change the expressions of p-CREB in protein level and CREB in mRNA level (all p>0.05).②The immunoreactive products of BDNF distributed in the cytoplasm, neucli and axons of pyramidal neurons within hippocampal CA1 subfield. The BDNF expressions in protein and mRNA levels were higher in 2VO group than in sham-operated group (p<0.05). Administration of ISA at the dose of 100mg/kg could markedly increase BDNF expression in protein and mRNA levels, compared to the 2VO group (both p<0.05). Dihydroergotoxine and ISA at the doses of 50mg/kg and 25mg/kg didn’t significantly change BDNF expression in protein and mRNA levels (all p>0.05).③The immunoreactive products of TrkB distributed in the cytoplasm, neucli and axons of pyramidal neurons within hippocampal CA1 subfield. The TrkB mRNA expression was higher in 2VO group than in sham-operated group (p <0.05). But the protein expression level didn’t change significantly in 2VO rats. Administration of ISA at three dosages and Dihydroergotoxine didn’t significantly change TrkB expression in protein levels and mRNA levels (all p>0.05)④The immunoreactive products of ChAT distributed in the cytoplasm and axons of pyramidal neurons within hippocampal CA1 subfield. The ChAT expression in protein and mRNA levels were lower in 2VO group than in sham-operated group (p<0.01). Administration of Dihydroergotoxine and ISA at the dose of 50 mg/kg and 100mg/kg increased the ChAT expressions in protein level (all p<0.01). ISA significantly increased ChAT mRNA expression at the dose of 100mg/kg, compared to 2VO group (p<0.05, p<0.01). Dihydroergotoxine and ISA at the dose of 50 mg/kg and 25mg/kg didn’t change ChAT mRNA expression markedly (all p>0.05).⑤The immunoreactive products of SYP surrounded the pyramidal neurons, showing the profile of the cell body and axon in the hippocampal CA1 region. Few immunoreactive products were observed in the cytoplasm and nucleu. The SYP expression in protein and mRNA levels were lower in 2VO group than in sham-operated group (p<0.05). Administration of Dihydroergotoxine and ISA at all doses increased the SYP expressions in protein level (all p<0.01). ISA increased SYP mRNA expression at the dose of ISA100mg/kg (p<0.01). Administration of Dihydroergotoxine and ISA at other doses didn’t markedly change the SYP expressions in mRNA level (all p >0.05).Conclusions:①The declines of cholinergic function and synaptic loss accounted for the cognition deficits in rats subjected to CCH.②ISA enhanced the activity of ChAT and expression of SYP via increasing BDNF expression, subsequently improved the synaptic transmission efficiency.③ISA improved the learning and memory through enhancing the cholinergic function and synaptic plasticity in hippocampus of CCH rats.Conclusions for the whole thesis:1. The present research exhibited two kinds of VD rat model. The rats with photochemical lesion or CCH both showed significant declines in spatial learning and memory. The neuronal loss existed in the parietal cortex and hippocampal CA1 subfield respectively in the above-mentioned two sorts of model rats. The further transmission electron microscopy indicated the pathological changes of synaptic ultrastructure in hippocampal CA1 subfield of 2VO rats.2. Compared to the photochemical lesion model, the 2VO rat model showed better stability, higher living rate and lower experimental costs. Most of all, it better mimiced the pathophysiologic process of VD in patients. So in the present research, we took use of the 2VO model to investigate the effects and mechanisms of ISA on rats with CCH.3. The research showed that the treatment with ISA was benefit for rats with CCH, as evidenced by administration of ISA could ameliorate the spatial learning and memory, reduce the neuronal loss and enhance the structural plasticity of hippocampal synapse in rats. ISA showed better effects at the dose of 100mg/kg.4. The research also indicated that the decline of cholinergic function and synaptic loss contributed to the cognition deficits in rats with CCH.5. BDNF contributed to the compensation mechanism. ISA could enhance the endogenous neuroprotection induced by CCH through increasing the expression of BDNF in mRNA and protein levels in hippocampal CA1 subfield. Up-regulating the expressions of SYP and ChAT, two downstream proteins of BDNF-TrkB might be the main mechanism underlying the effects of ISA. (1) Administration of ISA for 8 weeks could significantly increase the number of ChAT immunoreaction-positive cell per unit area, the percentage of immunoreaction-positive area and ChAT mRNA expression in hippocampal CA1 subfield. This result indicated that the enhancement of cholinergic function induced by ISA could potentially supply the substance basis to the synaptic plasticity. (2) Administration of ISA could significantly increase the SYP expression in mRNA and protein levels, subsequently improved the learning and memory via enhancing synaptic transmission efficiency.6. To conclude, ISA improved the learning and memory through enhancing the cholinergic function and protecting the synaptic structure in hippocampus of CCH rats.

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