| Vascular dementia(VD),one of the neurodegenerative diseases induced by cerebrovascular diseases,is characterized by severe cognitive impairment caused by reduction or block in cerebral blood flow(CBF).With the development of economic and the improvement of living standards,the incidence of cerebrovascular diseases has increased year by year,and these diseases have a tendency to occur among the young.VD has become the second most common type of dementia after Alzheimer's disease(AD),which brought huge economic and psychological burdens to patients and their family.When patients diagnosed with VD,they always missed the best time for the prevention and treatment at the initial stages.So on the basis of the research for VD,a broader concept,vascular cognitive impairment(VCI),has been put forward.VCI refers to a kind of syndrome that contains all forms of cognitive dysfunction caused by cerebrovascular diseases,from mild cognitive impairment to dementia.Studies found that cerebral ischemia/reperfusion(IR)contributed to progressing learning and memory impairment and hippocampal neuron damage,which is one of major causes of VCI.Therefore,researches on the pathogenesis and related treatments of VCI induced by cerebral IR have attracted more attention.Until now the underlying mechanisms of VCI have not been completely characterized.Studies found that cerebral IR causes oxidative stress which further damages hippocampal neurons,is a major mechanism of VCI.Brain tissues,especially hippocampus tissues,are sensitive to hypoxia-ischemia.Recent studies found that cerebral IR leads to the disorder of normal metabolic of neurons and increases the generation of reactive oxygen species(ROS),which beyonds the scavenging ability of neurons.Oxidative stress induced by cerebral IR breaks dynamic balance between endogenous oxidants and antioxidants and damages the neurons.Malondialdehyde(MDA),the product of lipid peroxidation,is a reliable marker of oxidative stress,which can reflect the severity of cellular damage.In addition,free radical scavengers,reduced glutathione(GSH),and superoxide dismutase(SOD)play a vital role in protecting brain tissues from oxidative damage.Researches found that the levels of MDA,GSH and the activity of SOD in brain tissues are abnormal after cerebral IR.Furthermore,the destruction of the normal structures of neurons,especially the destruction of the mitochondrial structure,further aggravates oxidative stress damage and forms the vicious circle.Nrf2-ARE pathway is the most important endogenous antioxidant stress pathway,which has protective effect on neurodegenerative diseases.Under normal physiological conditions,Nrf2 is inactive and unites with kelch-like ECH-associated protein 1(Keap1),located in the cytoplasm.In response to oxidative stress,Nrf2 is actived and dissociates from Keap1.Then Nrf2 translocates from the cytoplasm to the nucleus and binds to the antioxidant response element(ARE)to activate transcription of many Nrf2-ARE-related phase ? enzymes,such as heme oxygenase-1(HO-1)and NAD(P)H: quinine oxidoreductase 1(NQO1),which protects against oxidative stress damage.Previous studies found that the Nrf2 and NQO1 protein expressions in the hippocampus of mice with cognitive dysfunction induced by chronic cerebral hypoperfusion are abnormal.Oxidative stress damage in the brains of these mice induces hippocampal neuron apoptosis,which demonstrates that the Nrf2-ARE pathway may be closely related to the onset of VCI.Recent studies demonstrate that anti-oxidative stress therapies can delay or even reverse the development of VCI with learning and memory impairment through regulating the Nrf2-ARE pathway,which may be a novel therapy target of VCI.Astaxanthin(ATX),which is a naturally occurring carotenoid pigment,is widespread in marine organisms.ATX possesses excellent anti-antioxidant activity and can be transported across the blood-brain barrier(BBB).A previous study revealed that pre-treatment with ATX could decrease oxidative stress to protect cortical neurons and reduce infarct volume in focal cerebral IR of rats.ATX can improve cognitive function damaged by aluminum chloride through protecting hippocampal neurons.Moreover,ATX suppresses oxidative stress and reduces early brain injury in subarachnoid hemorrhage via activating of the Nrf2-ARE pathway.However,so far no study has examined the potential efficacy of ATX treatment on cognitive function after cerebral IR and the possible regulatory mechanism.The most common model used to replicate the pathology and behavior changes of VCI patients is bilateral common carotid arteries occlusion(BCCAO)model.Separation and clip the bilateral common carotid artery causes the acute decline in CBF,leading to acute cerebral ischemia of the animals.Rodents have abundant collateral circulation of the brain.Method of cutting the tail and bloodletting to lower blood pressure increases the stability of the model.The BCCAO model has low mortality.Animals after cerebral IR appear obvious behavior dysfunctions,such as learning and memory impairment.Histopathological study also showed that cerebral IR causes hippocampal neuron loss.This model has similar clinical and pathological features with cerebral ischemic stroke of VCI patients.It is an ideal animal model of VCI and suitable for exploring the pathogenesis and corresponding treatment for VCI.The mouse model of VCI induced by BCCAO was used in the present study to observe the changes of learning and memory behavior and morphology of hippocampal neurons;detect the changes of the ultrastructure of hippocampal neurons,oxidative stress parameters and expressions of apoptosis related proteins;examine the changes of Nrf2,HO-1 and NQO1 expressions in hippocampus of VCI mice after ATX treatment and further evalue the effect on the learning and memory behavior and oxidative stress related damage in hippocampus of VCI Nrf2+/+ and Nrf2-/-mice after ATX treatment.This study is to explore the influence of ATX on learning and memory function of mice after cerebral IR and the corresponding mechanism.It is hoped that the results could provide some experimental bases for the treatment of VCI.Part1 Astaxanthin ameliorates cognitive impairment and hippocampal neuron damage in cerebral ischemia/reperfusion miceObjective: To observe the changes of cognitive function and morphology of hippocampal neuron in cerebral IR mice after ATX treatment.Methods: Male ICR mice with BCCAO were used to establish cognitive impairment mice induced by cerebral IR.All mice were divided into sham operated(sham)group,model(VCI)group,ATX-treated sham operated(sham+ATX)group and ATX-treated model(VCI+ATX)group.After surgery,the mice received ATX(10 mg/kg/d)or an equal volume of vehicle by daily intragastric administration for 28 days.Morris water maze test and Nissel staining were adopted to analysis the behavioral changes of cognitive function and morphology of hippocampal neurons respectively.Results:1 Gerenal condition: After surgery,the gerenal condition of the mice in the sham group was good.The weight of these mice gradually increased during the dosing.During the 3 days postoperative,the gerenal condition of the mice in the VCI group was bad.The weight of these mice gradually decreased and the hair of these mice was without burnish.The condition bagan to improve gradually 3 days later.During the 2 days postoperative,the gerenal condition of the mice in the VCI+ATX group was similar with that of the mice in the VCI group.But this condition bagan to improve soon.The gerenal condition of the mice in the VCI+ATX group was better than that of the mice in the VCI group,and worse that of the mice in the sham group.The number of deaths was 3 in the VCI group and the mortality rate was 15.79% after surgey for 4 weeks.The number of deaths was 2 in the VCI+ATX group and the mortality rate was 11.11% after surgey for 4 weeks.No death was observed in the sham group and the sham+ATX group;2 Morris water maze testThe navigation test: There were significant differences among all of the groups(P<0.01)and all mice showed a progressive reduction in escape latency with training(P<0.01).On test days 2-5,mice of the VCI group exhibited significantly prolonged escape latency compared to mice in the sham group on the same day(P<0.01).On test days 3-5,mice in the VCI+ATX group showed significantly shortened escape latency compared to mice in the VCI group on the same day(Day 3: P < 0.05;Day 4,5: P<0.01).There were no significant differences in escape latency between the sham group and the sham+ATX group on the same day(P>0.05);The probe trail: There were significant differences among all of the groups in the number of times the mice crossed the platform(P<0.01).The number of times the mice crossed the platform and the time in target quadrant in the VCI group were less than those in the sham group(P<0.01).The number of times the mice crossed the platform and the time in target quadrant of mice in the VCI+ATX group obviously increased(P<0.01),but have not yet restored to the levels of mice in the sham group(P<0.01).There were no significant differences in the number of times the mice crossed the platform between the sham group and the sham+ATX group(P>0.05);The visible platform test: There were no significant differences in escape latency and average swimming speed among mice in all groups(P>0.05);3 Nissl staining: There were significant differences among all of the groups in the morphological structure and the number of surviving pyramidal neurons(P<0.01).In the sham group,aligned pyramidal neurons in sections of the CA1 and CA3 regions,arranged tightly and had normal cell microstructures.In the VCI group,the arrangement of surviving pyramidal neurons was loose and the neurons had abnormal structure.The number of surviving pyramidal neurons in the CA1 and CA3 regions was significantly decreased in the VCI group compared to neurons in the sham group(P<0.01).Treatment with ATX reversed the morphological damage and significantly rescued the pyramidal neuron loss(CA1: P<0.01;CA3: P<0.05).There was no significant difference in the morphological structure and the number of surviving pyramidal neurons in the CA1 and CA3 regions between the sham group and the sham+ATX group(P>0.05).Summery:1 Cerebral IR injuryed spatial learning and memory function of mice,leading to VCI.2 ATX ameliorated spatial learning and memory impairment in mice with cerebral IR.3 ATX had neuropertective effects on hippocampal neuron damage of mice induced by cerebral IR.Part2 The protective effects of astaxanthin on oxidative stress damage in the hippocampus of cerebral ischemia/reperfusion miceObjective: To observe the changes of oxidative stress parameters and neuron ultrastructure in the hippocampus,and to detect whether the hippocampal neuron apoptosis reduced in cerebral IR mice after ATX treatment.Methods: Male ICR mice with BCCAO were used to establish cognitive impairment mice induced by cerebral IR.All mice were divided into sham operated(sham)group,model(VCI)group,ATX-treated sham operated(sham+ATX)group and ATX-treated model(VCI+ATX)group.The changes of oxidative stress parameters in mice hippocampus were detected by biochemical methods.The changes in the ultrastructure of hippocampal neurons were analyzed by transmission electron microscopy(TEM).Western blot method was used to analyze the expression of apoptosis related proteins in hippocampus of mice.Results:1 Oxidative stress parameters: There were significant differences among all of the groups in the levels of MDA,GSH and the activity of SOD in the hippocampi(P<0.01).The level of MDA was significantly higher(P<0.01),and the level of GSH and the activity of SOD in the VCI group were markedly lower than those of the sham group(P<0.01).However,the VCI+ATX group showed a significant decrease in the level of MDA(P<0.01)and obvious increases in the concentration of GSH(P<0.05)as well as the activity of SOD(P<0.05)compared to the VCI group.Comparison of the levels of MDA,GSH and SOD in the hippocampus revealed no significant difference between the sham group and the sham+ATX group(P>0.05);2 TEM: The ultrastructure of hippocampal neurons from the mice in the sham group was basically normal,with round or oval nuclei,and well-defined and complete nuclear membranes.Clear and normal-shaped mitochondria were distributed in the cytoplasm of these neurons.The ultrastructure of hippocampal neurons from the mice in the VCI group,showed marked changes.The nuclei were irregular and the nuclear membranes were partially dissolved.The mitochondria were swollen,with degenerated vacuoles and fractured cristae.ATX reduced edema and organelle damage in the hippocampal neurons.The mitochondria were essentially normal,with only slight swelling and fracturing of cristae.There were no significant differences between the hippocampal neurons of mice in the sham+ATX group and the hippocampal neurons of mice in the sham group;3 Western blotGroup differences in the levels of Cyt C,cleaved Caspase-3,Bax and Bcl-2 proteins(P<0.01)were found in the hippocampi of all mice.Compared to the sham group,the expression of Cyt C,cleaved Caspase-3 and Bax in the VCI group increased markedly(P<0.01),whereas the expression of Bcl-2 decreased(P<0.01).Compared to the mice of VCI group,the expression of Cyt C,cleaved Caspase-3 and Bax in the mice of VCI+ATX group decreased(P<0.05),and the expression of Bcl-2 significantly increased(P<0.01).There was no significant difference in the levels of Cyt C,cleaved Caspase-3,Bax and Bcl-2 proteins between the sham group and the sham + ATX group(P>0.05).Summery:1 ATX may ameliorate cognitive impairment of mice with cerebral IR by its antioxidant effect.2 ATX alleviated damage to the ultrastructure of hippocampal neurons in cerebral IR mice.3 ATX has neuroprotective effects on reducing hippcampal neuron apoptosis through resistance to oxidative stress damage.Part3 Astaxanthin has neuroprotective effects on vascular cognitive impairment in cerebral ischemia/reperfusion mice via the Nrf2-ARE pathwayObjective: To observe whether the Nrf2-ARE pathway is involved in regulating neuroprotective effects on vascular cognitive impairment of mice.Methods: BCCAO method was adopted to establish the mouse model of VCI induced by cerebral IR.(1)Male ICR mice were divided into sham operated(sham)group,model(VCI)group,ATX-treated sham operated(sham+ATX)group and ATX-treated model(VCI+ATX)group.Real time PCR(RT-PCR)and Western blot method were used to analyze the expression of Nrf2,HO-1 and NQO1 expression in hippocampus of VCI mice after ATX treatment.(2)Male Nrf2+/+ and Nrf2-/-mice were divided into Nrf2+/+ model(Nrf2+/+ VCI)group,ATX-treated Nrf2+/+ model(Nrf2+/+ VCI+ATX)group,Nrf2-/-model(Nrf2-/-VCI)group and ATX-treated Nrf2-/-model(Nrf2-/-VCI+ATX)group.The effect on the cognitive function,hippocampal neuron loss,oxidative stress damage and ultrastructure in the hippocampus of VCI Nrf2-transgenic mice after ATX treatment were observed.Results:1 Gerenal condition of the Nrf2+/+ and Nrf2-/-mice: During the 3 days postoperative,the gerenal condition of the mice in the Nrf2+/+ VCI group was bad.The weight of these mice gradually decreased and the hair of these mice was without burnish.The condition bagan to improve gradually 3 days later.During the 2 days postoperative,the gerenal condition of the mice in the Nrf2+/+ VCI+ATX group was similar with that of the mice in the Nrf2+/+ VCI group.But this condition bagan to improve soon.The gerenal conditions of the mice in the Nrf2-/-VCI group and the Nrf2-/-VCI+ATX group were similar,which were worse than that of the mice in the sham group.The number of deaths was 2 in the Nrf2+/+ VCI group and the mortality rate was 14.29% after surgey for 4 weeks.The number of deaths was 1 in the Nrf2+/+ VCI+ATX group and the mortality rate was 7.70% after surgey for 4 weeks.The number of deaths was 4 in the Nrf2-/-VCI group and the mortality rate was 25% after surgey for 4 weeks.The number of deaths was 3 in the Nrf2-/-VCI+ATX group and the mortality rate was 20% after surgey for 4 weeks;2 RT-PCR: The relative expressions of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA in the mice of sham group,VCI group,sham+ATX group and VCI+ATX group has significant statistical differences(P<0.01).Compared to the sham group,the relative expressions of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA in the VCI group decreased markedly(P<0.01);the relative expressions of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA in the VCI+ATX group significantly increased(P<0.01).There was no significant difference between the sham group and the sham+ATX group(P>0.05);3 Western blot: There was no significant difference in the protein expressions of Nrf2,HO-1 and NQO1 in the mice of sham group,VCI group,sham+ATX group and VCI+ATX group(P<0.01).Compared to the sham group,the protein expressions of Nrf2,HO-1 and NQO1 in the VCI group decreased markedly(P<0.01);the protein expressions of Nrf2,HO-1 and NQO1 in the VCI+ATX group significantly increased(Nrf2,HO-1: P<0.05;NQO1: P<0.01).No significant difference was found between the sham group and the sham+ATX group(P>0.05);4 Morris water maze testThe acquisition experiments: There were significant differences among all of the groups(P<0.01)and all mice showed a progressive reduction in escape latency with training(P<0.01).On test days 2-5,mice of the Nrf2+/+ VCI+ATX group exhibited significantly prolonged escape latency compared to mice in the Nrf2+/+ VCI group on the same day(Day 2,3: P<0.05;Day 4,5: P<0.01).On test days 3-5,mice in the Nrf2-/-VCI group showed significantly shortened escape latency compared to mice in the Nrf2+/+ VCI group on the same day(P<0.05).However,compared to the Nrf2-/-VCI group,the escape latency of mice in the Nrf2-/-VCI+ATX group did not exhibited obvious difference on the same day(P>0.05);The probe trail: Compared to mice in the Nrf2+/+ VCI group,the number of times the mice crossed the platform and the time in target quadrant in the Nrf2+/+ VCI+ATX group significantly increased(P<0.01).The number of times the mice crossed the platform and the time in target quadrant of mice in the Nrf2-/-VCI group obviously decreased(P<0.01)compared to mice in the Nrf2+/+ VCI group.However,compared to the Nrf2-/-VCI group,the escape latency of mice in the Nrf2-/-VCI+ATX group did not exhibited obvious difference(P>0.05);The visible platform experiments: There were no significant differences in escape latency and average swimming speed among mice in all groups(P>0.05);5 Nissl staining: There were significant differences in the number of surviving pyramidal neurons in CA1 region of hippocampus among all of the groups(P<0.01).The hippocampal pyramidal neurons of CA1 region in the Nrf2+/+ VCI group were damaged and arranged loose.Compared to the Nrf2+/+ VCI group,this morphological damage was reversed and the number of surviving pyramidal neurons significantly increased in the Nrf2+/+ VCI+ATX group(P<0.01).Compared to the Nrf2+/+ VCI group,the morphological damage was more severe and the number of surviving pyramidal neurons significantly decreased in the Nrf2-/-VCI group(P<0.01).There were no significant differences in the hippocampal neurons of CA1 region between the Nrf2-/-VCI group and the Nrf2-/-VCI + ATX group(P>0.05);6 Oxidative stress parameters: The level of MDA was significantly decreased(P<0.01),and the level of GSH and the activity of SOD in the Nrf2+/+ VCI+ATX group were markedly increased,compared to the Nrf2+/+ VCI group(P<0.01).The Nrf2-/-VCI group showed a significant increase in the level of MDA(P<0.05)and obvious decreases in the concentration of GSH(P<0.05)as well as the activity of SOD(P<0.05)compared to the Nrf2+/+ VCI group.However,comparison of the levels of MDA,GSH and the activity of SOD in the hippocampus revealed no significant difference between the Nrf2-/-VCI group and the Nrf2-/-VCI+ATX group(P>0.05);7 TEM: The nuclei of hipppcampal neurons in the Nrf2+/+ VCI group were irregular and the nuclear membranes were partially dissolved.The mitochondria in this group were swollen with broken cristae.The ultrastructural damage in the Nrf2+/+ VCI+ATX mice was attenuated.Although the mitochondria of neurons were still slightly enlarged,the structural damage was significantly reduced.The organelles were severely damaged and nuclear chromatin condensed to the nuclear membranes in the Nrf2-/-VCI mice compared to the Nrf2+/+ VCI mice.The mitochondria were severely swollen,vacuoles degenerated,and the cristae broke.However,there were no significant differences in ultrastructure in the hippocampal neurons between the Nrf2-/-VCI group and the Nrf2-/-VCI+ATX group.Summery:1 ATX can activate the Nrf2-ARE pathway and increased the expression of Nrf2,HO-1 and NQO1 in the hippocampus of cerebral IR mice.2 ATX ameliorated cognitive function and reduced oxidative stress damage through activating the Nrf2-ARE pathway of mice with cerebral IR.3 ATX has neuroprotective effect on VCI mice by activating the Nrf2-ARE pathway.Conclusions:1 Mice wih cerebral IR has spatial learning and memory dysfunction and hippocampal neuron damage,which are ideal models to study the pathogenesis and related treatment of VCI.2 ATX has neuroprotective effect on learning and memory dysfunction and hippocampal neuron damage of mice with VCI.3 ATX ameliorates the ultrastructural damage and apoptosis of hippocampal neurons of mice with VCI through suppressing oxidative stress.4 ATX ameliorates the cognitive function and reduces oxidative stress damage via activating the Nrf2-ARE pathway in the hippocampus of mice with VCI,which may be the neuroprotective mechanism of ATX. |
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