| Background and purposeAlzheimer's disease(AD)is a multifactorial,progressive,irreversible neurological degenerative disease that accounts for about 70%of all cases of dementia.It is the most common type of dementia due to its onset.The rate is closely related to age,so it is also called Alzheimer's.So far,about 47 million people worldwide have been diagnosed with AD.With the increasing number of elderly people in the world,it is expected that the number of patients with AD will reach 130 million by 2050,which will cause a heavy economic burden and social impact.AD has become one of the most urgent public health issues in the world.Although a large number of researchers have made great progress in the pathogenesis of AD and drug development,no effective treatment plan and drugs have been found to interfere with the progress of the disease.Epidemiological investigations have shown that the risk factors that cause AD are irreversible,such as metabolism,age,gender,and genetic predisposition.Therefore,the discovery of controllable risk factors that affect the occurrence and development of AD,the search for biological indicators and early diagnosis of AD,and early detection and early intervention are of great significance to delaying the AD process.Elevated plasma Hcy levels are manifested as hyperhomocystinemia(HHcy).With the continuous research on HCY,it is found that HHcy is closely related to the occurrence of many age-related diseases,such as cardiovascular disease,osteoporosis,stroke,Alzheimer's disease,and Parkinson's disease.Epidemiological studies and analysis of a large number of clinical data show that the increase in plasma HCY concentration is positively correlated with the occurrence of AD.HHcy is considered to be a strong and independent risk factor for AD,but the specific mechanism of action between the two is still unclear.The discovery of neurogenesis in the adult mammalian brain overturns the long-held belief that the adult brain is incapable of producing new neurons.Research suggests that adult mammalian neurogenesis exists only in the subventricular zone(SVZ)of the lateral ventricular wall and the subgranular zone(SGZ)of the hippocampal dentate gyrus.Neurogenesis in the central system,especially in the hippocampus,plays an important role in the structural plasticity of the nervous system and the maintenance of the network,and is conducive to the important role of information storage in learning and memory.Studies have shown that there is a strong correlation between impaired neurogenesis and impaired cognitive function.It is unclear how HCY affects neurogenesis in the hippocampus.This study mainly explores the effect of hyperhomocysteinemia on the cognitive function of SD rats,and analyzes its effect on neurogenesis in the hippocampal DG region of rats,and preliminary studies on the relationship between hyperhomocysteinemia and AD There are potential molecular mechanisms among them.Method1.37? physiological saline dissolves homocysteine powder,and is prepared into a solution with a final concentration of 400 ?g/ml.Rats in the Hcy group were injected with homocysteine solution via the tail vein at a dose of 400 ?g/kg/d for 14 consecutive days.Rats in the control group were injected with equal volume of normal saline through the tail vein.2.The content of plasma homocysteine in rats was detected by ELISA to determine whether the rat model of hyperhomocysteinemia was successfully constructed.After successful modeling,the Barnes maze experiment was carried out first,followed by the Morris water maze experiment.Rats were sacrificed 24 hours after the end of the behavioral experiment for the detection of various indicators.3.Using Morris water maze and Barnes maze two animal behavioral experiments to study the relationship between hyperhomocysteinemia and cognitive function.Before Hcy injection,all rats were subjected to the Morris water maze spatial positioning cruise test for 5 consecutive days.In order to make the rats have similar spatial learning and memory capabilities,a total of 50 rats that could find the hidden platform within 30 seconds were selected randomly.For the Hcy group and the control group,25 animals in each group.Fourteen days after the injection of Hcy,the Morris water maze space exploration experiment was conducted the next day to detect memory retention in rats.The Barnes maze was used to test the learning and memory abilities of rats.After modeling,two groups of rats were used to learn and memorize the spatial location of the avoidance box on the Barnes maze for 5 consecutive days,and the incubation period of the rats was recorded.The sixth day is the test day,recording the rat incubation period and the number of erroneous caves.4.To further clarify the causes of cognitive dysfunction in rats caused by hyperhomocysteinemia,ELISA was used to detect the content of A ? in the cortex and hippocampus.5.After the behavioral experiment was completed,5 rats were randomly selected from each group,and intraperitoneal injection of 6%chloral hydrate(0.6ml/100g)was performed on a stereotactic brain device for long-term enhanced hippocampal(LTP)detection.The diameter of the drill hole of the skull is about 1.5mm.Slowly insert the concentric circle stimulation electrode into the hippocampal schaffer collateral/combined fiber pathway(4.2mm behind the breech,3.8mm to the right of the midline,and 3.5mm below the skull).The recording electrode is located in the hippocampus.Radiation layer in CA1 area(3.4mm behind the bregma,2.5mm to the right of the midline,and 3mm below the skull).Record excitatory post-synaptic potentials(fEPSPs)in the radiation layer of CA1 area.Give test stimulation for at least 30 minutes before conditioned stimulation.After obtaining stable basal transmission,use high frequency stimulation to induce LTP of fEPSPs and record for 60 minutes.6.To investigate the effect of hyperhomocysteinemia on hippocampal synapses in rats,transmission electron microscopy was used to observe the ultrastructure of synapses after Hcy injection,and immunoblot technique was used to detect synapse-related proteins PSD93,PSD95,GluR1,GluR2,Expression of GluN2A and GluN2B.7.To investigate the effect of hyperhomocysteinemia on the dendritic spines of hippocampal CA1 neurons in rats,Golgi staining technique was used after Hcy injection to observe the density of dendritic spines in hippocampal CA1 neurons of rats.8.In order to investigate the effect of brain-derived neuroinfluencing factor on nerve regeneration in hippocampus,the expression of brain-derived neuroinfluencing factor(BDNF)in rat hippocampus was detected by immunoblotting after Hcy injection.At the same time,BDNF was supplemented in vitro,and a total of four groups were set as control group,Hcy group,BDNF group and K252a group.Rats were anesthetized with 10%chloral hydrate(300mg/kg),fixed to the brain stereotaxic instrument,the skull was opened 4.0mm behind the bregma and 2.0mm left/right beside the midline,and holes were drilled on both sides of the hippocampus CA3.The BDNF group was injected with 2 ? 1 BDNF(50 ng/mL),the K252a group was injected with 2 u1 BDNF(50 ng/mL)and the 2ul TrkB specific inhibitor K252a(0.2 ? M),and the Hcy and control rats were injected with sterile saline.Bone wax seals the hole and sutures the wound.Post-operative injection of 100,000 units of penicillin prevents wound infection.9.To explore the effect of hyperhomocysteinemia on neuronal regeneration in the hippocampal DG region of rats,thymidine analogs,5'bromodeoxyuracil nucleoside(BrdU),were used after Hcy injection to mark the neonatal hippocampal GD region Neurons.BrdU powder is dissolved in physiological saline at 37? and the final concentration is 10 mg/ml.The solution is protected from light when it is configured and is used now.The rats were injected with BrdU solution intraperitoneally for 4 consecutive days,the injection volume was 100 mg/kg,twice a day,the time interval was 8 hours,and the material was taken 24 hours after the last injection.The number of BrdU positive cells in SGZ and GCL regions of rat hippocampus was detected by immunohistochemical staining technique.10.To explore the effect of hyperhomocysteinemia on mature neurons in the hippocampus of rats,immunohistochemical staining technique was used after Hcy injection to count NeuN positive cells in the CA1 and CA3 regions of the hippocampus of rats.11.In order to detect neuronal damage,Nissl staining technique was used after Hcy injection to count the number of neurons in hippocampal CA1 and CA3 regions.12.All experimental data results were statistically analyzed using SPSS 21.0 software,with P<0.05 as the significance test standard.Result1.The results of ELISA detection of plasma homocysteine in rats showed that the plasma Hcy content in Hcy group was significantly higher than that in control group(P<0.01).2.Morris water maze results showed that the escape latency of rats injected with homocysteine was significantly longer than that of the control group(P<0.01).The space exploration experiment was performed after the platform was removed,and the results showed that the stay time in the second quadrant and the number of times of crossing the original platform in the model group were significantly reduced compared with the control group(P<0.01).3.The Barnes labyrinth experiment results showed that after 3 days of training,the latency of the control group rats was significantly shorter than that of the Hcy group(P<0.05).During the test,the number of incorrect burrowing in the Hcy group was significantly higher than that in the control group,and the escape latency was prolonged(P<0.05).4.ELISA method was used to detect the content of A ? in the cortex and hippocampus of rats.The results showed that the content of A ? in the cortex and hippocampus of Hcy group was significantly higher than that of the control group(P<0.05).5.The results of electrophysiological experiments showed that the LTP of CA3-CA1 loop in the Hcy group was significantly lower than that in the control group(P<0.001).6.Golgi staining results showed that the dendritic spine density of hippocampal CA1 neurons in the model group was significantly lower than that in the control group(P<0.05).7.Transmission electron microscopy and immunoblot results show that hyperhomocysteinemia can cause reduction of the number of synaptic vesicles in the presynaptic membrane,blurred synaptic gaps,uneven electron density of the pre-and post-synaptic membranes in the hippocampus of rats,and damage Synaptic structural integrity.The expression of synapse-related proteins PSD93,PSD95,GluR1,GluR2,GluN2A,and GluN2B in the hippocampus decreased significantly(P<0.01).8.Immunoblotting results showed that compared with the control group,the expression of BDNF protein in the hippocampus of the homocysteine group was significantly reduced(P<0.05).9.Brdu immunohistochemical staining showed that compared with the control group,hyperhomocysteinemia could lead to a significant reduction in the number of BrdU-positive cells in the SGZ and GCL regions of the rat hippocampus.After BDNF supplementation,the number of BrdU-positive cells in the SGZ and GCL regions of the hippocampus increased significantly compared with the model group.After K252a was added to block the binding of BDNF and TrkB receptor,the protective effect of BDNF on newborn neurons was significantly inhibited.10.NeuN immunohistochemical staining showed that the number of NeuN positive cells in the hippocampal CA1 and CA3 regions of the model group rats was significantly reduced compared with the control group.After adding BDNF,the number of NeuN positive cells in hippocampal CA1 and CA3 areas of BDNF group increased significantly compared with the model group.After K252a was added to block the binding of BDNF to the receptor TrkB,the protective effect of BDNF on mature neurons was significantly inhibited.11.Nissl staining results show that compared with the control group,the increase in plasma homocysteine levels can cause the number of Nissl bodies in the hippocampal CA1 and CA3 regions to decrease,the arrangement is disordered,and the Nissl borders are blurred or even disappeared.The number of Nissl bodies in the hippocampal CA1 and CA3 regions of rats after BDNF supplementation was significantly increased compared with the model group,and the Nissl bodies were relatively aligned.After K252a was added to block the binding of BDNF to the receptor TrkB,the protective effect of BDNF on neurons was significantly inhibited.ConclusionHomocysteine causes a decrease in BDNF protein levels in the hippocampus and impairs neurogenesis in the hippocampus.This may be a potential one cause of Alzheimer's-like cognitive dysfunction caused by homocysteine in SD rats. |
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