| Vascular dementia(VaD),which accounts for about 17-20%of all dementia patients,is the second most common form of dementia after Alzheimer's disease(AD)and is prevalent in the elderly population.VaD can be caused by reduced blood supply,which may or may not be related to stroke.Underdiagnosis of VaD,lack of treatment options,increasing life expectancy,and a steadily growing population with risk factors such as high blood pressure,heart disease,diabetes,metabolic syndrome,and stroke call for the development of a treatment for VaD.There are currently no FDA-approved treatment options for VaD,and some studies aim to develop a good animal model of VaD as a first step in exploring treatment options.In this study,the most promising bilateral carotid artery stenosis(BCAS)model was used as the animal model for the study of VaD.Pituitary adenylate cyclase-activated polypeptide(PACAP)is a neuropeptide originally isolated from the hypothalamus of sheep brain.PACAP has been shown to affect a variety of physiological activities including neurotransmission,neuroprotection,vasodilation,neuroregulation,and immunosuppression.Some studies have also shown that PACAP can effectively protect neurons from ischemic injury.PACAP and its receptors have also been shown to be involved in learning and memory processes.However,there are few studies on the promotion of synaptic plasticity and improvement of cognitive ability by PACAP,especially the possible role of PACAP-induced neuroprotection on cognitive impairment caused by chronic hypoperfusion(CCH)remains unclear.In this study,the neuroprotective effect of PACAP was confirmed in vivo and in vitro through the construction of VaD animal model and oxygen-glucose deprivation(OGD)neuron cell model,and studying of pathological changes,behavioral changes and cognition-related proteins in the brain tissue of the model,as well as the basic research of the pathway related to the effect of PACAP in neuronal cell lines.The important role of the PACAP-Sirt3-BDNF pathway in synaptic integrity was first elucidated.PACAP can inhibit cell apoptosis and protect the cognitive dysfunction caused by CCH by enhancing synaptic plasticity through Sirt3.Part ? The expression levels of PACAP,Sirt3 and synaptic proteins were significantly decreased in VaD modelObjective:To establish a model of VaD and detect the changes in the expression levels of PACAP,Sirt3,BDNF,PSD-95 and other proteins in this model,so as to clarify the role of PACAP,Sirt3,synaptic proteins and other key proteins in this model.Methods:1.Selection of experimental animals:SPF male C57BL/6 mice from Beijing Vital River Laboratory Animal Technology Co.,Ltd were selected,with age 9 to 11 weeks and a body weight of 23 to 26 g.The mice were housed in a constant temperature and humidity environment with a room temperature of24±2°C and a relative humidity of 50%-60%,and artificially given a light-dark12-hour alternating cycle of illumination to ensure the enough feeding and clean water source.2.Establishment of bilateral common carotid artery stenosis(BCAS)model:VaD model was established by BCAS surgery.Chloral hydrate(5%,350mg/kg)was used for intraperitoneal anesthesia in the model mice.The carotid sheath was separated by sharp forceps,and the vessels were separated medially from the common carotid artery(which had a large white vagus nerve on the lateral side,and the respiration of mice stopped rapidly after accidental injury).The lower edge of the common carotid artery bifurcation(the location of internal carotid artery and external carotid artery bifurcation)was hung,the common carotid artery was lifted,and the 0.18mm spring coil was quickly rotated to the blood vessel to ensure that the blood vessel was smooth without tortuously shaped.Then apply the spring to trap to the contralateral common carotid artery in the same manner after an interval of 30 minutes.After that,the skin and fascia were sutured quickly and placed in an incubator until they were fully awake.In the sham operation group,the steps were the same as above except for the spring coil.The cognitive behavior test was performed 30 days after the BCAS operation to complete the model construction.3.Laser speckle was used to detect the changes of cerebral blood flow(CBF)in VaD mice model:the anesthetized mice were incised along the midline of the skull skin to fully expose the skull in the prone position.The CBF of the mice was detected by laser speckle,monitored continuously for 3-5 minutes,and the blood flow value was recorded after the value stabilized.Laser speckle blood flow images were recorded for identification of ROI.In these ROIs,the mean blood flow index was calculated in real time.The changes of CBF in mice were detected before and 30 minutes after operation.4.The cognitive level of VaD mouse model was tested by novel recognition test(NOR test):mice were placed in a square open field(side length40 cm,height 40 cm)in a dimly lit room.Before the test,the mice were placed in an empty environment without objects for 2 days,and then stood for 10minutes to familiarize themselves with the experimental environment.On the third day of the test,the mice were exposed to two identical objects(Object A and B),freely explored object A and B for 10 minutes(the training phase),and then returned to the cage.After 24 hours,one of the two identical objects was randomly replaced before the test.The mice were then put back into an open field to explore freely for five minutes.The color,shape,and size of the replacement objects were completely different from the objects in the training phase.To avoid olfactory cues,apply 75%alcohol wipes to clean the site and objects between tests.Object preference is measured as time spent exploring new objects×100/time spent exploring new and familiar things.Ethvision XT software was used to track and record animal tracks.5.Nissl staining was used to detect the apoptosis of cerebral cortex neurons in VaD mouse model:30 days after BCAS,the brain tissues of the mice were fixed,embedded in paraffin,and coronally cut into 6?m thick wax slices and placed on glass slides.After dewaxing and gradient alcohol hydration,the brain sections were immersed in toluidine blue dye solution and incubated in an oven at 56°C for 25 minutes.The brain slices were washed in water with distilled water for 5 minutes.The decolorization was performed by gradient dehydration in 75%,95%and 100%ethanol for 3 seconds each.After decolorization,slices were placed in xylene for 5 minutes,twice.Finally,the sections are sealed with neutral balsam.Tissue target area was selected for 400X imaging using the Eclipse CI-L microscope.Image-Pro Plus 6.0 analysis software was used to measure the number of neurons in each field of view,called A,corresponding tissue area(mm2)called B,and neuron density calculated as n/mm2(A/B).6.Morphological changes of neurons and mitochondria in VaD mice model were observed by transmission electron microscopy:in order to observe the ultrastructure of cerebral cortex neurons,1 mm3volume of cortical brain tissue was taken and fixed with a mixture of 2%paraformaldehyde and 2.5%glutaraldehyde for 2 hours.After dehydration,the tissue was embedded in Epon resin.Ultra-thin sections of 70 nm thickness were cut and stained with lead citrate and uranium acetate.Morphological changes of neurons and their mitochondria were observed under transmission electron microscopy.7.The expression of proteins was detected by Western blot:including total protein extraction,protein concentration determination,electrophoresis,membrane transfection,blocking,antibody incubation,membrane scanning,and analysis.8.Immunofluorescence staining was used to observe the changes in the expression of PSD-95 synaptic protein.After the paraffin sections were dewaxed,tissue sections were hydrated with gradient alcohol,and the antigen was repaired by microwave immersion with sodium citrate solution at 92-96?for 30 minutes.Brain tissue was incubated with 10%goat serum at room temperature for 1 hour.After removing the excess serum,the primary rabbit anti-PSD-95 antibody was added,and then placed at 4?overnight.The next day,the tissue sections were cleaned with PBS for 3 times,5 minutes each time.Then the tissues were incubated with Alexa FluorTM 488 donkey anti-rabbit antibody for 1 hour at room temperature and protected from light.After that wash them 3 times with PBS,5 minutes each time.The tissue was sealed with an Antifade Solution containing DAPI,stored at 4°C and protected from light,and then photographed with a fluorescence microscope.Image J software was used for semi-quantitative analysis.9.Statistical methods:the results are presented as the means±SD.The normal test was performed by Shapiro-Wilk test.Statistical differences between groups were evaluated by two-tailed unpaired Student's t-test.One-way ANOVA analysis was used to determine the significance of multiple groups.Mann-Whitney U test was used for nonparametric data.Values of p<0.05 were accepted as significant using SPSS version 23.0.All experiments were repeated at least 3 times.Results:1.After BCAS,CBF decreased significantly:CBF decreased 34.5%in BCAS mice compared to pre-operation,P<0.001.2.Thirty days after operation,the mice in the BCAS group had cognitive impairment:the time of exploring new things in the BCAS group was 24.80%lower than that in the sham group(P<0.01).3.Nissl staining of VaD brain tissue showed no obvious neuronal apoptosis.4.The ultrastructure of neurons in VaD mice was changed:the abnormality of neurons,nuclear pyknosis,mitochondria swelling and the number of cristae decreased were observed by transmission electron microscopy.5.PACAP,Sirt3 protein expression and synaptic plasticity were decreased in brain tissues of VaD mice:Western Blot results showed that the level of PACAP protein in brain tissues of VaD group was decreased(58.84%decrease vs.Sham,P<0.01),and the level of PAC1 protein was not significantly decreased.In addition,the protein levels of Sirt3,BDNF and PSD-95 were all decreased in the VaD group,but not in the SYN group.Immunofluorescence staining showed that the synaptic protein PSD-95 was decreased in the BCAS group compared with the sham group,suggesting decreased synaptic function and plasticity.Summary:After 30 days of BCAS modeling,cognitive impairment occurred.VaD mouse model was successfully constructed.Ultrastructure of brain tissue was changed in this model,and the expression levels of PACAP,Sirt3 and synaptic proteins were significantly reduced.Part ? PACAP enhances synaptic plasticity and plays an anti-apoptotic role by acting on Sirt3Objective:The oxygen-glucose deprivation(OGD)model of cells was established to simulate the hypoperfusion condition of brain tissue,and the expression changes of Sirt3,apoptosis related proteins and synaptic proteins after intervention were observed.The neurons were pretreated with PACAP1-38 to clarify its protective effect on OGD.Lenti-virus was used to overexpress or knock down Sirt3 to explore the changes in the expression of synaptic proteins and to elucidate the mechanism of the protective effect of PACAP in neurons.Methods:1.Cell culture and establishment of OGD model:HT22 hippocampal neuron cell line was cultured in DMEM medium.OGD model:cells growing to80-90%were taken and washed with OGD buffer for 3 times,then removed OGD buffer and readd OGD buffer.The culture plate was placed in a hypoxic workstation,and the mixture of 5%CO2,94%N2and 1%O2 was fed into the hypoxic workstation and the cells were cultured at 37?.2.Lentiviral construction of stable cell lines:The exogenous mouse Sirt3cDNA sequence was subcloned into the Lenti-CMV-GFP vector to overexpress Sirt3.A short sequence of 19 nucleotides was constructed and Sirt3 site 764 was targeted to Omics Link small hairpin RNA(sh RNA)expression clones to knock down Sirt3 expression.Successfully transfected cells express green fluorescence.Viral particles transfected cells:The lenti-virus was involved in transfecting the cells and puromycin was used to screen the untransfected cells.3.Application of PACAP1-38 for HT22 cells:PACAP1-38 was prepared with sterile water into 0.1mg/ml stock solution,diluted with fresh DMEM medium to prepare different drug concentrations,and co-cultured with HT22cells for 24 hours.4.CCK-8 kit was used to measure the cell viability of HT22 cells:HT22cells treated with OGD were inoculated into 96-well plates(inoculated cell suspension was 100?l/well,5000 cells/well),and 10?l CCK solution was added to each well.After the plates were placed in the incubator for 1 hour,the absorbance at 450nm was measured with a microplate analyzer.5.The expression of proteins was detected by Western blot:including total protein extraction,protein concentration determination,electrophoresis,membrane transfection,blocking,antibody incubation,membrane scanning,and analysis.6.Cell immunofluorescence staining was used to detect the expression of PSD-95:the cells inoculated on the coated slides were washed 3 times with PBS,the residual medium was removed,and then fixed with 4%paraformaldehyde for 10 minutes.After fixation,the cells were washed with PBS for 3 times for 5minutes.Then they were incubated with 10%goat serum at room temperature for 1 hour.After removing the excess serum,anti-PSD-95 primary antibody was added,and incubated at 4?overnight.The next day,the cells were cleaned with PBS for 3 times,5 minutes each time.The slices were incubated with Alexa FluorTM 488 donkey anti-rabbit antibody for 1 hour at room temperature and protected from light.Then wash them 3 times with PBS,5 minutes each time.Then get the slices out from the orifice plate and place it upside down on the glass slide containing the antifade solution with DAPI,stored at 4°C and protected from light,and then photographed with a fluorescence microscope.Image J software was used for semi-quantitative analysis.7.Statistical method:the results are presented as the means±SD.The normal test was performed by Shapiro-Wilk test.Statistical differences between groups were evaluated by two-tailed unpaired Student's t-test.One-way ANOVA analysis was used to determine the significance of multiple groups.Mann-Whitney U test was used for nonparametric data.Values of p<0.05 were accepted as significant using SPSS version 23.0.All experiments were repeated at least 3 times.Results:1.PACAP1-38 can promote the protein expression of PACAP,Sirt3 and BDNF in HT22 cells:PACAP1-38 cultured at 50n M and 100n M significantly increased the level of PACAP(435.10%increase vs.0n M,P<0.05;1479.00%increase vs.0n M,P<0.05).Sirt3 and BDNF expression were increased in cells with 100n M PACAP1-38 pretreated(42.41%increase vs.0n M,P<0.05;Increase vs.0n M,P<0.05).There was no significant change in PSD-95 protein.2.PACAP could improve the viability of HT22 cells under OGD:cell viability of HT22 cells began to decline 2 hours after OGD treatment,which decreased to 75.89%(P<0.001)at OGD 4 hours and 48.71%(P<0.001)at OGD6 hours.200n M PACAP1-38 pretreatment increased cell viability by 39.57%(P<0.05)after OGD 6 hours.3.PACAP1-38 can enhance synaptic plasticity and inhibit cell apoptosis under the condition of OGD:the protein levels of Sirt3,BDNF and PSD-95decreased after OGD 6 hours,and the protein levels of Sirt3,BDNF and PSD-95 in cells pretreated with PACAP1-38 for 24 hours were increased after OGD6 hours treatment compared with those in the group without PACAP1-38.The anti-apoptotic proteins Bcl-2 and the ratio of Bcl-2/Bax decreased after OGD 6hours treatment,and recovered after PACAP1-38 pretreatment.4.Sirt3 can regulate BDNF and synaptic plasticity:it was found that BDNF and PSD-95 protein levels changed consistently with the expression change of Sirt3 through lentiviral transfection which knocking down or overexpressing Sirt3,that is,the expression of BDNF and PSD-95 decreased after knockdown of Sirt3,and PACAP1-38 could not improve the effect of knockdown of Sirt3.The expression of BDNF and PSD-95 increased after Sirt3 overexpression.Summary:PACAP1-38 can promote the protein expression of PACAP,Sirt3 and BDNF in HT22 cells,improve the cell viability of HT22 cells under the condition of OGD,enhance the synaptic plasticity of HT22 cells,and inhibit the apoptosis induced by OGD.Part ? PACAP can effectively improve cognitive impairment of VaD by improving synaptic plasticityObjective:The model of VaD was established,and PACAP1-38 was used to intervene the mice model to observe whether it could improve the expression level of synaptic plasticity related proteins and improve the cognitive impairment of VaD.Methods:1.Selection of experimental animals:SPFmale C57BL/6 mice from Beijing Vital River Laboratory Animal Technology Co.,Ltd were selected,with age 9 to 11 weeks and a body weight of 23 to 26 g.The mice were housed in a constant temperature and humidity environment with a room temperature of 24±2°C and a relative humidity of50%-60%,and artificially given a light-dark 12-hour alternating cycle of illumination to ensure the enough feeding and clean water source.2.The VaD model establishment and grouping:the model mice of BCAS after 30 days were divided into two groups:BCAS+NS group(normal saline treatment 3?l/nostril)and BCAS+PACAP group(PACAP1-38 treatment with0.1?g/?l,3?l/nostril).The drug was given at the same time 5 times per week for4 weeks.3.The cognitive level of VaD mouse model was tested by novel recognition test(NOR test):mice were placed in a square open field(side length 40 cm,height 40 cm)in a dimly lit room.Before the test,the mice were placed in an empty environment without objects for 2 days,and then stood for 10 minutes to familiarize themselves with the experimental environment.On the third day of the test,the mice were exposed to two identical objects(Object A and B),freely explored object A and B for 10 minutes(the training phase),and then returned to the cage.After 24 hours,one of the two identical objects was randomly replaced before the test.The mice were then put back into an open field to explore freely for five minutes.The color,shape,and size of the replacement objects were completely different from the objects in the training phase.To avoid olfactory cues,apply 75%alcohol wipes to clean the site and objects between tests.Object preference is measured as time spent exploring new objects×100/time spent exploring new and familiar things.Ethvision XT software was used to track and record animal tracks.4.The expression of proteins was detected by Western blot:including total protein extraction,protein concentration determination,electrophoresis,membrane transfection,blocking,antibody incubation,membrane scanning,and analysis.5.Statistical methods:the results are presented as the means±SD.The normal test was performed by Shapiro-Wilk test.Statistical differences between groups were evaluated by two-tailed unpaired Student's t-test.One-way ANOVA analysis was used to determine the significance of multiple groups.Mann-Whitney U test was used for nonparametric data.Values of p<0.05 were accepted as significant using SPSS version 23.0.All experiments were repeated at least 3 times.Results:1.PACAP1-38 can improve the expression levels of PACAP,Sirt3,BDNF and synaptic plasticity in VaD mice:the protein levels of PACAP,PAC1,Sirt3,BDNF and PSD-95 in BCAS+PACAP group were all increased compared with BCAS+NS group.2.PACAP1-38 can improve the cognitive impairment of VaD mice:the time of exploring new things in BCAS+NS group was 24.80%lower than that in BCAS+PACAP group(P<0.01).Summary:Nasal administration of PACAP1-38 increased the expression levels of PACAP,Sirt3,and BDNF and synaptic plasticity in VaD brain tissue,and improved cognitive impairment in VaD mice.Conclusion:1.The expression of PACAP,Sirt3,BDNF,PSD-95 which were cognition-related proteins in the brain tissues of VaD mice was decreased.This suggests the potential as a diagnostic marker for PACAP and Sirt3.2.PACAP can increase the activity of neurons after OGD,inhibit their apoptosis,and improve the levels of PACAP,Sirt3,BDNF,PSD-95 proteins.3.PACAP regulates the level of synaptic protein and improves synaptic plasticity through Sirt3.4.PACAP improved cognitive impairment in VaD mice by regulating the PACAP-Sirt3-BDNF pathway. |
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