| Vascular dementia(VD)is a clinical syndrome of acquired cognitive dysfunction caused by various cerebrovascular factors.With the advent of the aging of the global population,the incidence of cerebrovascular disease is increasing year by year,and VD has become the second leading cause of cognitive dysfunction after Alzheimer’s disease(AD).However,up to now,the pathogenesis of VD has not been fully clarified.The treatment focuses on the control of related cardiovascular and cerebrovascular risk factors and accompanying symptoms,and lacks effective therapeutic drugs.Therefore,to explore the pathogenesis of VD and to seek effective drugs to prevent and treat VD has become more important and urgent.Estrogen is a steroid hormone,not only involved in affecting the female reproductive system,but also regulating glycolipid metabolism and bone metabolism and protecting the cardiovascular system.The brain is one of the target organs of estrogen,and estrogen subjects are widely distributed in the hippocampus,cerebral cortical layer,hypothalamus,basal forebrain and other cognitively related areas.Epidemiological data show that the incidence of stroke in premenopause women is significantly lower than in men of the same age,but there is no significant difference after post-menopause.Recent studies have found that post-menopause women accepted estrogen replacement therapy have a significantly lower incidence of dementia,suggesting that estrogen may play an important role in the development of dementia.These observations suggest that estrogen may be a promising new treatment for VD.In the central nervous system,the hippocampus make an important role in learning memory,information processing and signal transmission.However,the CA1 region of hippocampus is the most vulnerable area of the brain in the low perfusion state.When the hippocampus is damaged by ischemia hypoxia,multiple signal transductive paths in the cell are abnormal,apoptosis and autophagy are activated at the same time,thus accelerating the death of neurons.Wnt/β-Catenin signaling pathways play an important role in the development of the central nervous system in mammals and are the key ways in regulating cell growth,proliferation and differentiation.But so far,there has been little literature on whether estrogen can affect the learning and memory ability of VD rats by regulating the apoptosis,autophagy,and Wnt/β-Catenin signaling pathogenes in the vascular dementia model.Based on the above research present situation,adult female SD rats were randomly divided into 4 groups(sham,model,estrogen early and estrogen later treatment)and received sham surgery or bilateral ovariectomy(OVX)and permanent occlusion of bilateral common carotid arteries(BCCAO).The early treatment group received daily intraperitoneal injections of 17β-estradiol(100 μg/kg/day)for 8 weeks starting the day just after BCCAO.The later treatment group was administered the same starting 1 week after BCCAO.Learning and memory functions were assessed using the Morris water maze.Morphological changes within the hippocampal CA1 region were observed by hematoxylin/eosin staining and electron microscopy.Expression of proteins associated with apoptosis and autophagy and signaling were detected by immunohistochemical staining and Western blot.On this basis,the correlation between the neuroprotective effect of ertrogen and the activation of Wnt/β-catenin signal transduction pathway was discussed,providing a more comprehensive theoretical basis for the mechanism of estrogen to improve memory and cognitive impairment.Part One Establishment of ovariectomized vascular dementia rat model and effects of estrogen on cognitive function of ovariectomized vascular dementia ratsObjective:To observe the effects of estrogen on the cognitive function of VD rats.Methods:1.A total of 48 healthy SD rats were randomly divided into four groups:sham group,model group,E2-early group and E2-later group,with 12 rats in each group.2.The rats in sham group were removed only a small amount of adipose tissue around the ovaries,while the rats in other three groups were removed both sides of the ovaries.One week after OVX surgery,the rats in sham group received BCCAO sham surgery,and the others received BCCAO to prepare VD models.3.The rats in E2-early group received intraperitoneal injection of 17β-estradiol(E2)from the first day after BCCAO surgery,100 μg/kg/day,the solvent was sesame oil.While the rats in E2-later group received intraperitoneal injection of E2 one week after BCCAO surgery.The rats in sham surgical group and the model group received the same amount of sesame oil celiac injection.Each group of rats had intraperitoneal injections once a day for a total of 8 weeks.4.Just from the day after the last dose,the rats were tested for Morris Water Maze behavior to assess the rats’ spatial learning and memory abilities.The first 5 days are Place navigation test and the 6th day is Spatial probe test.Results:1.Place navigation testEscape latency,i.e.the time from entry to stage,was used as an indicator to evaluate the spatial learning ability of SD rats.With the increase of training days,the average escape latency of the four groups of rats gradually shortened,and statistics showed significant differences in escape latency at various points in time in the same group.At the first day of navigation experiment,there was no significant difference in the escape latency of the four groups(P>0.05).On day 2-5 of the navigation experiment,the escape latency of rats in the model group was significantly higher than that in the sham-operated group(day 2,P<0.05;3-5 days,P<0.01).After estrogen intervention,the escape latency of rats in the E2-early group was significantly shorter than that in the model group(Day 3 and Day 4,P<0.05;On day 5,P<0.01).The escape latency of E2-later group was significantly shorter than that of model group on day 5(P<0.05).2.Spatial probe testOn the 6th day of the experiment,the platform was removed to record the percentage of total time spent of rats in the original platform quadrant within 120 seconds(target quadrant time percentage),the percentage of the original platform quadrant distance to the total distance(target quadrant distance percentage),and the number of times it crossed the location of the original platform(number of stage crossings)as the evaluation index of spatial memory ability of rats.The percentage of time in the target quadrant and the percentage of distance in the model group were significantly shortened,indicating that BCCAO caused the memory deficit of rats,while the percentage of time in the target quadrant and the percentage of distance in the E2-Early group were significantly increased compared with the model group.The number of cross-platform crossing in model group was significantly decreased compared with sham-operated group(P<0.01).Compared with model group,the number of cross-platform crossing in E2-Early group was significantly increased(P<0.01).Although the percentage of target quadrant time,percentage of target quadrant swimming distance and number of cross-platform crossing in E2-later group were higher than those in model group,the differences were not statistically significant.3.Changes of swimming trajectories of rats in four groupsWhen the rats first entered the water,they swam randomly to look for the underwater platform,and the swimming track was mostly on the edge of the water maze.With the increase of training days,the swimming trajectories of sham-operated group and estrogen treatment group gradually become a trend type,especially the sham-operated group was even close to the linear model.However,until the end of training,the search mode in model group rats was still mostly edge mode,reflecting the model group rats appeared obvious cognitive impairment.Conclusion:1.OVX combined with BCCAO can successfully establish the castrated VD rat model,which can simulate the learning and memory dysfunction caused by chronic cerebral hypoperfusion.2.Estrogen therapy can effectively improve the learning and memory ability of VD rats,and early estrogen intervention is more effective than later estrogen intervention.Part Two Effects of estrogen on hippocampal histomorphology in rats with vascular dementiaObjective:To observe the histopathological changes of hippocampal neurons of VD rats and the effect of estrogen administration time on the morphology of hippocampal neurons.Methods:After the behavioral test,the rats were anesthetized and blood samples were collected from the heart.Serum estradiol values were determined by ELISA method.Morphological changes of neurons in the hippocampal CA1 region were observed by light microscope and transmission electron microscopy.Results:1.Observed results of serum estradiol concentrationSerum estrogen concentration in model group was significantly lower than that in sham-operated group and estrogen treatment group.There was no significant difference in serum estrogen concentration between estrogen treatment group and sham-operated group.2.Observed characteristics of HE staining in the hippocampal CA1 region of ratsThe pyramidal neurons within the hippocampal CA1 region of the sham-operated group were morphologically normal,arranged and in order,and exhibited round and plump nuclei,clear nucleoli and cytoplasm.In contrast,in the model group,the hippocampal neurons were arranged loosely and disordered,significantly decreased in number,and mostly fusiform or polygon in sharp,with irregular nuclei.Some of those neurons exhibited karyopyknosis and hyperchromatic cytoplasm,and the nucleoli were blurred or had disappeared entirely.Estrogen intervention protected animals from such morphological changes.These improvements of hippocampal morphology were more significant in the E2-early treatment group than that of the later treatment group.3.Ultrastructural changes of neurons in hippocampal CA1 region of ratsThe ultrastructure of neurons within the hippocampal CA1 region was normal in the sham-operated group,with regular and plump oval nuclei,a clear double-layer nuclear membrane,uniform arrangement of chromatin,and a normal number of organelles of typical morphology in the cytoplasm.In contrast,in the model group,the nuclei were deformed and nucleoli have shrunken,nuclear membranes were dissolved,nuclear chromatin increased and agglomerated into lumps,cytoplasmic organelles significantly reduced and deformed,the remaining mitochondria swollen and vacuolated,and the rough endoplasmic reticulum expanded and broken.In response to estrogen treatment,the ultrastructural damage of nuclei and organelles was significantly reduced,the shape of the nuclei appeared normal,and the organelles were clear.Remarkably,compared to the E2-early treatment group,the ultrastructural damage of neurons in the E2-later treatment group were still substantial.Conclusion:There were obvious histopathological changes in neurons in the hippocampal CA1 area of VD rats.After the intervention of estrogen,the damage of hippocampal neurons in VD rats was relatively improved.Estrogen may act in a way that treats time dependence.Part Three Effects of estrogen on apoptosis and autophagy related protein expression in hippocampal neurons of rats with vascular dementiaObjective:To observe the apoptosis-related proteins in the hippocampus of VD rats:Bc1-2,Bax and caspase-3;and autophagy related proteins:LC3Ⅱ,Beclinl expression level.To observe the regulation effect of estrogen on neuronal cell apoptosis and autophagy,and to explore its neuroprotective mechanism.Methods:1.Animal grouping and intervention mode were the same as the first part.2.The expression of Bcl-2,Bax,caspase-3 and Beclinl positive cells in the hippocampal area of rats in each group was observed by Immunohistochemistry method.The expression of LC3Ⅱ positive cells in hippocampal area of rats was observed by immunofluorescence staining.The dynamic changes of Bcl-2,Bax,caspase-3,Beclinl,LC3Ⅱ protein expression in the hippocampal tissues of rats were detected by Western Blot.Results:1 The expressions of Bcl-2,Bax,caspase-3 and Beclinl in rat hippocampus were detected by immunohistochemistry.1.1 Under light microscope,Bcl-2 immunopositive products were brown yellow particles,and the cytoplasm and nucleus of neurons were localized.Compared with the sham-operated group,the IOD/area value of Bcl-2 in hippocampal CA1 region of model group was significantly decreased(P<0.05).Compared with the model group,the IOD/area value of Bcl-2 in E2-early group was significantly higher(P<0.05),while the IOD/area value of Bcl-2 in E2-later group was higher than that in model group,but there was no statistically significant difference.1.2 Under light microscope,Bax immunopositive products were brown yellow particles,mainly located in neuronal cytoplasm.The IOD/area value of Bax in hippocampal CA1 region of model group was significantly higher than that of sham-operated group(P<0.05).Compared with model group,the IOD/area value of Bax in E2-early group was significantly decreased(P<0.05),while the IOD/area value of Bax in E2-later group was lower than that in model group,but there was no statistical difference.1.3 Under light microscope,the caspase-3 immunopositive products were brown particles,and the cytoplasm and nucleus of the neurons were localized.Compared with sham-operated group,the IOD/area value of caspase-3 in hippocampal CA1 region in model group was significantly increased(P<0.05).Compared with the model group,the IOD/area value of caspase-3 in the E2-early group was significantly decreased(P<0.05),while the IOD/area value of caspase-3 in the E2-later group was lower than that in model group,but there was no statistical difference.1.4 Under light microscope,Beclinl immunopositive products were brown yellow particles,which were localized to the neuronal cytoplasm.Compared with sham-operated group,the IOD/area value of Beclinl in hippocampal CA1 region in model group was significantly increased(P<0.01).Compared with the model group,the IOD/area value of Beclinl in E2-early and E2-later groups was significantly decreased,but there was no statistical difference between E2-early group and E2-later group.2 The expressions of LC3II in rat hippocampus were observed by immuno-fluorescence.LC3Ⅱ protein was labeled with yellow-green fluorescence in this experiment.Fluorescence microscopy showed that the immunofluorescence expression of neurons in the sham-operated group was weak,and the immunofluorescence intensity of LC3Ⅱ in the model group was significantly increased(P<0.01).Compared with model group,LC3Ⅱ immuno-fluorescence expression intensity in E2-early group and E2-later group was significantly decreased,but there was no statistical difference between E2-early group and E2-later group.3 Western blot results3.1 Expression of Bcl-2,Bax,caspase-3,LC3II and Beclinl in the hippocampus of model group rats.3.1.1 Compared with the sham-operated group,the protein expression of Bcl-2 in the model group was significantly decreased(P<0.05),while the protein expression of Bax and caspase-3 in the model group were significantly increased.3.1.2 Compared with the sham-operated group,the expression levels of LC3Ⅱprotein and Beclinl protein in the model group were significantly increased.3.2 Effects of estrogen on protein expressions of Bcl-2,Bax,caspase-3,LC3Ⅱand Beclinl in hippocampus of VD rats.3.2.1 Compared with the model group,the protein expression of Bcl-2 was significantly increased,the protein expression of Bax was significantly decreased,and the protein expression of caspase-3 was significantly decreased in E2-Early and E2-Later groups,with statistical differences.3.2.2 Compared with the model group,LC3Ⅱ protein expression was significantly decreased in the E2-early group and E2-later group,and Beclinl protein expression was significantly decreased in the E2-early group and E2-later group.Conclusion:The apoptosis of hippocampal neurons in VD rats was increased and autophagy was activated,leading to damage and death of hippocampal neurons.Estrogen can ameliorate hippocampal injury by reducing neuronal apoptosis and down-regulating autophagy activation.Part Four Effects of estrogen on Wnt/β-catenin signaling transduction pathway in rats with vascular dementiaObjective:To observe the effect of estrogen on Wnt/β-catenin signaling transduction pathway in VD rats,and to discuss its molecular biological mechanism.Methods:1.Forty-eight healthy adult female SD rats were randomly divided into four groups:sham-operated group,model group,E2 group,and E2+DKK1 group.2.Only a small amount of adipose tissue around ovary was removed in the sham-operated group,and the other three groups were treated with OVX.One week after OVX,the model group,the E2 group and the E2+DKK1 group received BCCAO surgery to prepare the VD model,while the sham-operated group received BCCAO sham surgery.Rats in the E2+DKK1 group received lateral ventricle catheterization on the first day after BCCAO surgery.3.Rats in the E2 group and the E2+DKK1 group received intraperitoneal injection of 17β-estradiol,100 μg/kg/day,respectively,from the first day after BCCAO surgery.Rats in the sham-operated group and the model group received intraperitoneal injection of the same amount of sesame oil,respectively.Once a day for a total of 7 days.Recombinant human DKK1 protein is used to block the Wnt/β-catenin signal transduction pathway.Rats in the E2+DKK1 group received lateral ventricle injection of DKK1 10 μl at a concentration of 1 μg/ul on the 1 and 4 days after BCCAO surgery.4.The samples were collected 7 days after BCCAO surgery,and the morphological changes of neurons in hippocampal CA1 region were observed by HE staining and transmission electron microscopy.The protein expressions of β-catenin,Cyclin D1 and GSK-3β in hippocampal tissues were detected by immunohistochemistry and Western blot.Results:1.Observed characteristics of HE staining in the hippocampal CA1 region of ratsThe neurons within the hippocampal CA1 region of the sham-operated group were morphologically normal,arranged and in order,and exhibited round and plump nuclei,clear nucleoli and cytoplasm.In contrast,in the model group,the hippocampal neurons were arranged disordered,and mostly fusiform or polygon in sharp,with irregular nuclei.Some of those neurons exhibited karyopyknosis and hyperchromatic cytoplasm,and the nucleoli had disappeared entirely.Estrogen intervention protected animals from such morphological changes.Neurons in hippocampal CA1 region of rats in E2+DKK1 group were loosely arranged and disorderly,most of them were vague in shape,with irregular nuclei,deep staining and pyknosis,and a halo was found around the cytoplasm,with large cell spacing.2.Ultrastructural changes of neurons in hippocampal CA1 region of ratsThe ultrastructure of neurons within the hippocampal CA1 region was normal in the sham-operated group,with regular and plump oval nuclei,a clear double-layer nuclear membrane,uniform arrangement of chromatin,and a normal number of organelles of typical morphology in the cytoplasm.In contrast,in the model group,the nuclei were deformed and nucleoli have shrunken,nuclear membranes were dissolved,nuclear chromatin increased and agglomerated into lumps,cytoplasmic organelles significantly reduced and deformed,the remaining mitochondria swollen and vacuolated,and the rough endoplasmic reticulum expanded and broken.In response to estrogen treatment,the ultrastructural damage of nuclei and organelles was significantly reduced,the shape of the nuclei appeared normal,and the organelles were clear.Neurons in hippocampal CA1 region of rats in E2+DKK1 group were deformed,with dissolved and ruptured nuclear membrane.Nuclear heterochromatin increased and agglomerated into lumps,cytoplasmic organelles significantly reduced,glycogen granules and mitochondria disappeared,the remaining endoplasmic reticulum and Golgy body expanded significantly.3 The expressions of β-catenin,Cyclin D1 and GSK-3β in rat hippocampus were detected by immunohistochemistry.3.1 The β-catenin immunopositive products are shown as brown-yellow particles localized to the cytoplasm of neurons under light microscope.The IOD/area value of β-catenin in hippocampal CA1 region in model group was significantly lower than that in sham-operated group(P<0.05).After estrogen treatment,the IOD/area value of β-catenin in the E2 group was significantly higher than that in the model group(P<0.05).However,after the administration of Wnt pathway inhibitor,β-catenin in the E2+DKK1 group was slightly higher than that in the model group,but the difference was not statistically significant.The IOD/area value of the E2+DKK1 group was significantly lower than that in the E2 group(P<0.05).3.2 The GSK-3β immunopositive products are shown as brown-yellow particles localized to the neuronal cytoplasm under light microscope.The IOD/area value of GSK-3β in hippocampal CA1 region of model group was significantly higher than that of sham-operated group(P<0.01).After estrogen treatment,the IOD/area value of GSK-3β in the E2 group was significantly lower than that in the model group(P<0.01).However,the IOD/area value of GSK-3 β in the E2+DKK1 group was lower than that in the model group(P>0.05),and the IOD/area value of the E2+DKK1 group was significantly higher than that in the E2 group(P<0.05).3.3 The Cyclin D1 immunopositive products are shown as brown-yellow particles localized to the neuronal cytoplasm under light microscope.The IOD/area value of Cyclin D1 in hippocampus CA1 in model group was significantly lower than that in sham-operated group(P<0.01).After estrogen treatment,the IOD/area value of Cyclin D1 in the E2 group was significantly increased compared with that in the model group(P<0.01),while the IOD/area value of Cyclin D1 in the E2+DKK1 group was slightly increased compared with that in the model group(P>0.05),and the IOD/area value of the E2+DKK1 group was significantly decreased compared with that in the E2 group(P<0.01).4 Western blot results4.1 Model group:compared with the sham-operated group,the protein expressions of β-catenin and Cyclin D1 in the hippocampus of rats were significantly decreased,and the protein expression of GSK-3β was significantly increased(P<0.01).4.2 E2 group:compared with model group,after estrogen intervention,this trend was significantly reversed,the protein expressions of β-catenin and Cyclin D1 were significantly increased,and the protein expression of GSK-3βwas significantly decreased(P<0.01).4.3 E2+DKK1 group:compared with estrogen treatment alone,the protein expressions of β-catenin and Cyclin D1 in rats were significantly decreased,and the protein expression of GSK-3β was significantly increased(P<0.05).Conclusion:Estrogen therapy can reduce the damage of hippocampal neurons in VD rats.The administration of the Wnt pathway inhibitor DKK1 significantly weakened the protective effect of estrogen,suggesting that estrogen may play its role by activating the Wnt/β-catenin signaling pathway.Part Five Effect of estrogen replacement therapy on postoperative cognitive function in surgical menopausalObjective:To explore the effect of estrogen replacement therapy on postoperative cognitive function in surgical menopausal.Methods:42 cases(all pre-menopausal)who underwent hysterectomy and bilateral ovariectomy due to benign lesions were selected from our hospital during January 2019 to January 2020,aged 48～54 years old.22 cases as the experimental group were given single estrogen therapy for 6 months;blank control group(n=20),did not receive hormone therapy.Serum estradiol level and cognitive function were compared between the two groups 6 months after treatment.Results:After treatment,the serum estradiol level of the experimental group was significantly increased compared with the control group(P<0.05),and the MoCA score was significantly increased compared with the control group(P<0.05).Conclusion:Estrogen replacement therapy can improve the cognitive function of postoperative in surgical menopausal. |
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