Electroacupuncture Rescues Cognitive Impairment In The APP/PS1Transgenic Mice Through Promotion Of Neurogenesis

【Background】Alzheimer’s disease (AD) is one of the most commonneurodegenerative diseases with the main clinical manifestations of dementia,memory loss, personality changes and language barriers. AD is not only withinsidious onset, progressive symptoms and severe interference with patients’daily lives, but also becomes more aggravating and presents a younger trend.According to the2000American census report, approximately4.5million peoplewere afflicted from Alzheimer’s disease, while in China the latest survey reportedan AD prevalence rate of2.12%over the age of60,3.00%over the age of65and4.69%over the age of70. Confronted with such a large quantity of peoplesuffering from Alzheimer’s disease, currently available AD treatments are stilllimited with poor curative effect. Therefore, seeking for an effective and safe wayfor treating Alzheimer’s disease is urgent.Electroacupuncture (EA), as one traditional Chinese medicine treatment bystimulating certain acupoint, could induce significant neuroprotective effect in various central nervous system diseases, as well as improve neuroethology defect.0ur previous studies have confirmed that the EA treatment could induce the rapidand delayed tolerance to focal cerebral ischemia in animal models and improveneurological function scores after stroke. We also proved that electroacupunctureis an effective way for treating+Gz-induced loss of consciousness. Due to a largenumber of domestic and foreign researches, electroacupuncture plays animportant role in promoting neurogenesis at the damage parts after brain injuryand this could be one underlying mechanism of EA neuroprotective effect.However, whether electroacupuncture treatment improves behavioralperformance and promotes neurogenesis in Alzheimer’s disease is still unclear.The pathology of Alzheimer’s disease generally accepted is characterized bythe abundant β-amyloid plaques abnormal deposition，neurofibrillary tangles, andselective neuron loss. The accumulation of β-amyloid peptides plays the mostimportant role in the pathogenesis of AD. Aβ1-42, the metabolite of amyloidprecursor protein (APP) and presenilin-1(PS1) gene mutation, is a crucialcomposition of β-amyloid plaques. The neurotoxicity of Aβ1-42also mediatesneuronal death in the brain. Neurogenesis, defined as a process of generatingfunctional neurons from neural stem cells or neural progenitor cells under certaininduction, has been proved to occur in the adult central nervous system and playimportant parts in adult brain function and recovery. The adult neurogenesiseffect is significantly suppressed in AD patients under the effects of aging as wellas accumulation of β-amyloid plaques, which in a sense aggravates the incidenceof dementia. Brain derived neurotrophic factor (BDNF) is one kind of proteinsthat serves a major role in central nervous system neurogenesis, differentiationand survival. It is involved in axonal plasticity, as well as the regulation ofneuronal differentiation and survival, neuroprotection and neurogenesis after neuronal injury.In the present study, we verified, including pathology and neuroethology,that electroacupuncture has a therapeutic effect on Alzheimer’s disease throughreducing the abnormal deposition of β-amyloid plaque, up-regulating BDNFexpression and promoting the neurogenesis. Thus, we provide a novel and simpleclinical treatment for Alzheimer’s disease. In this experiment, APP/PS1doubletransgenic mice model was used to simulation Alzheimer’s disease for systematicstudy.Experiment IConstruction and identification of Alzheimer’s disease double transgenicmice.Objective: To construct and identify the Alzheimer’s disease double transgenicmice model.Methods: Two-month-old C57mice with amyloid precursor protein (APP) andpresenilin-1(PS1) gene mutations were purchased from BEIJING HFKBIOSCIENCE CO., LTD. Two-month-old healthy C57BL/6J mice were providedby Experiment Animal Center of the Fourth Military Medical University.1)Morris water maze test was conducted among the two groups of animals in2-month,7-month and10-month respectively to investigate the change of spatiallearning and memory function.2）Immunohistochemistry double staining wasconducted in2-month,7-month and10-month old respectively to detect thechange of Aβ deposition with8mice each group. With the combination of thesetwo aspects, we identify the Alzheimer’s disease double transgenic mice modelfrom pathology and neuroethology.Results: There are no significantly differences of spatial learning and memory performance between Alzheimer’s disease double transgenic mice and wild-typeC57mice in2-month old mice. At7-month time point, the performance of spatiallearning and memory function in Alzheimer’s disease double transgenic micewere damaged more severely than wild-type C57mice (P<0.05). And suchneurobehavioral damage in the10-month old mice was further aggravated. At thesame time, the result of immunohistochemistry double staining was consistentwith the neuroethology results. There were no significantly differences of Aβdeposition in cortex and hippocampus between Alzheimer’s disease doubletransgenic mice and wild-type C57mice in2-month old group. Aβ deposition inAlzheimer’s disease double transgenic mice was increased in7-month and10-month groups. All shown that spatial learning and memory deficits andabnormal deposition of Aβ in Alzheimer’s disease double transgenic mice werebeginning at7-month old and the deficits were aggravated in10-month group.Conclusion: The model of Alzheimer’s disease double transgenic mice wassuccessfully constructed and could be used for the further study of the treatmentof Alzheimer’s disease.Experiment IIThe effect of EA on pathology and neuroethology of Alzheimer’s diseasedouble transgenic miceObjective: To investigate the effect of electroacupuncture stimulation on Aβ deposition and neuroethology of Alzheimer’s disease double transgenic mice.Methods: Grouped:48male AD mice were randomly divided into two groups:EA treatment group (APP+EA group) and control group (APP group)(n=24).48healthy male C57mice were randomly divided into two groups: EA treatmentgroup (Con+EA group) and control group (Con group)(n=24).1) To investigate EA stimulation on spatial learning and memory function of AD mice. EAtreatment was performed at the acupoint “Baihui (GV20)” with an intensity of1mA and frequency of15HZ for four weeks (30min/day,5day/week). DuringEA treatment, the rectal temperature of all animals was maintained at37.0±0.5℃and inhaled oxygen by facemask at a flow rate of1L/min. Neuroethology wasdetected the day after EA stimulation. The methods were the same withexperiment I.2) To investigate the effect of EA stimulation on Aβ deposition ofAD mice. The immunohistochemistry double staining, Elisa and Western blotwere conducted after water maze test to examine the change of Aβ depositionafter EA stimulation.Results:1) Water maze test showed that the spatial learning and memoryfunction were significantly improved in APP+EA group,with the extension oftarget quadrant time and the shorten of escape latency comparing with APP group.At the same time, compared with Con group, a slight improvement of spatiallearning and memory function were also detected in Con+EA group (P<0.05).The neurobehavioral performances in APP and APP+EA group were damagedmore severely than Con and Con+EA group.2）Immunohistochemistry doublestaining showed that Aβ expression in the neurons cytoplasm in the hippocampusand cortical regions. The expression level of Aβ in APP+EA group was lowerthan APP group, and the same change was found in wild-type C57mice. Theresult of Elisa and Western blot were consistent with immunohistochemistrydouble staining (P<0.05).Conclusion: EA stimulation has a therapeutic effect on AD mice throughimproving neurological behavior and decreasing Aβ deposition. Experiment IIIThe mechanism underlying the therapeutic effect of EA stimulation onAlzheimer’s diseaseObjective: To research the potential mechanism of the therapeutic effect of EAon Alzheimer’s disease.Method:1）The effect of EA on the expression of BDNF in central nervoussystem. After the Morris water maze test, each group of mice were perfused withparaformaldehyde and the brains were harvested for frozen sections.Immunohistochemistry double staining was conducted to observe co-localizationphenomenon of BDNF and neurons. At the same time, fresh brain tissue ofhippocampus and cortex was harvested and extracted for Western blot analysis todetect the expression of BDNF.2） The effect of EA on the number ofneurogenesis in central nervous system. Mice in each group were intraperitonealinjection of5-bromodeoxyuridine (BrdU), which is a kind of DNA marker tolabel newborn neurons,100mg/kg/day for7days.28days after the last injectionimmunohistochemistry double staining was conducted and the newborn neuronswere labeled.Results:1）Immunohistochemistry double staining showed that there wereco-localization phenomenon of BDNF and neurons. Western blot result indicatedthat compared with APP group，the level of BDNF in APP+EA group wassignificantly increased (P<0.05). Meanwhile, compared with Con group, thelevel of BDNF in Con+EA group was also significantly increased (P<0.05).2）Compared with APP group，the number of neurogenesis in APP+EA group wassignificantly increased (P<0.05). Compared with Con group, the number ofneurogenesis in Con+EA group was also significantly increased (P<0.05).Conclusion: The effect of EA on AD mice was through the promotion of brain expression of BDNF and neurogenesis.SummaryOur study indicated that EA simulatating on “Baihui” could significantlyimprove cognitive function in Alzheimer’s disease double transgenic mice. Themechanism may be related to reducing the deposition of β-like amylase,increasing the expression of BDNF and promoting neurogenesis. All of thisprovided a new treatment method and theorefical basis for the use of EA in theclinical treatment of Alzheimer’s disease.