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Optogenetics-induced activation of glutamate receptors improves memory function in mice with Alzheimer’s disease

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Neural Regeneration Research 2019年第12期14卷 2147-2155页

作者： Ke-Wei Wang Xiao-Lin Ye Ting Huang Xi-Fei Yang Liang-Yu Zou Department of Neurology Shenzhen People’s Hospital (First Affiliated Hospital of Southern University of Science and Technology) Second Clinical College Jinan University Shenzhen Guangdong Province China Key Laboratory of Modern Toxicology of Shenzhen Shenzhen Center for Disease Control and Prevention Shenzhen Guangdong Province China

Optogenetics is a combination of optics and genetics technology that can be used to activate or inhibit specific cells in tissues. It has been used to treat Parkinson’s disease, epilepsy and neurological diseases, but rarely Alzheimer’s disease. Adeno-associated virus carrying the CaMK promoter driving the optogenetic channelrhodopsin-2 (CHR2) gene (or without the CHR2 gene, as control) was injected into the bilateral dentate gyri, followed by repeated intrahippocampal injections of soluble low-molecular-weight amyloid-β1–42 peptide (Aβ1–42). Subsequently, the region was stimulated with a 473 nm laser (1–3 ms, 10 Hz, 5 minutes). The novel object recognition test was conducted to test memory function in mice. Immunohistochemical staining was performed to analyze the numbers of NeuN and synapsin Ia/b-positive cells in the hippocampus. Western blot assay was carried out to analyze the expression levels of glial fibrillary acidic protein, NeuN, synapsin Ia/b, metabotropic glutamate receptor-1a (mGluR-1a), mGluR-5, N-methyl-D-aspartate receptor subunit NR1, glutamate receptor 2, interleukin-1β, interleukin-6 and interleukin-10. Optogenetic stimulation improved working and short-term memory in mice with Alzheimer’s disease. This neuroprotective effect was associated with increased expression of NR1, glutamate receptor 2 and mGluR-5 in the hippocampus, and decreased expression of glial fibrillary acidic protein and interleukin-6. Our results show that optogenetics can be used to regulate the neuronal-glial network to ameliorate memory functions in mice with Alzheimer’s disease. The study was approved by the Animal Resources Committee of Jinan University, China (approval No. LL-KT-2011134) on February 28, 2011.

关键词： nerve regeneration Alzheimer's disease amyloid-β1-42 dentate gyrus channelrhodopsin-2 glutamate receptors memory neuroinflammation novel object recognition immunohistochemistry western blot assay neural regeneration

Early constraint-induced movement therapy affects behavior and neuronal plasticity in ischemia-injured rat brains

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Neural Regeneration Research 2019年第5期14卷 775-782页

作者： Xi-Hua Liu Hong-Yan Bi Jie Cao Shuo Ren Shou-Wei Yue Department of Physical Medicine & Rehabilitation Affiliated Hospital of Shandong Traditional Chinese Medicine University Maternal and Child Health Development Research Center Shandong Maternal and Child Health Hospital Department of Physical Medicine & Rehabilitation Qilu Hospital Shandong University

Constraint-induced movement therapy is an effective rehabilitative training technique used to improve the restoration of impaired upper extremity movement after stroke. However, whether constraint-induced movement therapy is more effective than conventional rehabilitation in acute or sub-acute stroke remains controversial. The aim of the present study was to identify the optimal time to start constraint-induced movement therapy after ischemic stroke and to explore the mechanisms by which constraint-induced movement therapy leads to post-stroke recovery. Sixty-four adult male Sprague-Dawley rats were randomly divided into four groups: sham-surgery group, cerebral ischemia/reperfusion group, early constraint-induced movement therapy group, and late constraint-induced movement therapy group. Rat models of left middle cerebral artery occlusion were established according to the Zea Longa line embolism method. Constraint-induced movement therapy was conducted starting on day 1 or day 14 in the early constraint-induced movement therapy and late constraint-induced movement therapy groups, respectively. To explore the effect of each intervention time on neuromotor function, behavioral function was assessed using a balance beam walking test before surgery and at 8 and 21 days after surgery. The expression levels of brain-derived neurotrophic factor, nerve growth factor and Nogo receptor were evaluated using real time-polymerase chain reaction and western blot assay to assess the effect of each intervention time. The results showed that the behavioral score was significantly lower in the early constraint-induced movement therapy group than in the cerebral ischemia/reperfusion and late constraint-induced movement therapy groups at 8 days. At 21 days, the scores had significantly decreased in the early constraint-induced movement therapy and late constraint-induced movement therapy groups. At 8 days, only mild pyknosis appeared in neurons of the ischemic penumbra in the early constraint-induced movement therapy group, which w

关键词： nerve regeneration ischemic stroke rehabilitation constraint-induced movement therapy nerve growth factors functional recovery neuronal plasticity real time-polymerase chain reaction western blot assay rats neural regeneration

Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

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Science China Chemistry 1994年第6期37卷 691-700,769页

作者：汪国顺罗文捷潘惟钧丁明孝翟中和 Department of Biology Peking University Beijing 100871 PRC

Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.

关键词： chromosomal telomere nuclear matrix lamina Southwestern blot ESI (Electron Spectro-scopic Imaging).

Expression and localization of absent in melanoma 2 in the injured spinal cord

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Neural Regeneration Research 2019年第3期14卷 542-552页

作者： Sai-Nan Wang Xue-Yan Guo Jie Tang Shu-Qin Ding Lin Shen Rui Wang Shan-Feng Ma Jian-Guo Hu He-Zuo Lü Clinical Laboratory the First Affiliated Hospital of Bengbu Medical College Anhui Key Laboratory of Tissue Transplantation the First Affiliated Hospital of Bengbu Medical College Department of Immunology Bengbu Medical College and Anhui Key Laboratory of Infection and Immunity at Bengbu Medical College

In traumatic brain injury, absent in melanoma 2(AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day(post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+(neurons), GFAP+(astrocytes), CNPase+(oligodendrocytes) and CD11 b+(microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+(leukocytes) and CD68+(activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.

关键词： nerve regeneration spinal cord injury absent in melanoma 2 spatio-temporal expression neurons astrocytes oligodendrocytes infiltrated leukocytes activated microglia western blot assay immunohistochemistry neural regeneration

Change of MicroRNA-134,CREB and p-CREB expression in epileptic rat

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Asian Pacific Journal of Tropical Medicine 2015年第4期8卷 292-298页

作者： Yan Zhu Cheng-Shan Li Yuan-Ye Wang Sheng-Nian Zhou Department of ICU(Jining medical college affiliated hospital) Department of Neurology Qilu Hospital of Shandong University Brain Science Research Institute Shandong University

Objective:To To investigate the changes of MicroRNA-134,CREB and p-CREB expression in epileptic rat brains in order to elucidate the molecular mechanisms of epilepsy,providing new ideas for clinical treatment.Methods:Sixty-four Spraque-Dawley(SD)rats were divided into groups randomly,including control group,six hours after seizure group,24-hour group,threeday group,one-week group,two-week group,four-week group,and eight-week group.All groups were placed under a pilocarpine-induced epilepsy model except the control group,and all rats were decapitated in different points of time.Brain specimens were taken for quantitative PCR experiments,immunohistochemistry and Western blot experiments.The results of the epilepsy model groups and the control group were compared.Results:There were no significant differences between the six hours after seizure group,the 24-hour group and the control group about the MicroRNA-134 levels.MicroRNA-134 in the hippocampus tissue of the three-day group significantly reduced compared with the control group;same result was observed with the one-week,two-week,four-week and eight-week groups.The CREB and p-CREB levels in the three-day group's rat hippocampus significantly increased compared with the control group;and the high levels of CREB and p-CREB were constantly maintained in the one-week,two-week,four-week and eight-week groups.Conclusions:The MicroRNA-134 level of the epileptic rat hippocampus is significantly lower than normal after three days,and continues to maintain a low level:while CREB and p-CREB levels are rsignificantly increased after three days,and continue to remain at a high level.MicroRNA-134 plays a role in inhibiting synaptic plasticity by inhibiting CREB and p-CREB expressions.

关键词： MicroRNA-134 CREB p-CREB Quantitative PCR Western blot Immunohistochemistry

Study of penetration mechanism of labrasol on rabbit cornea by Ussing chamber, RT-PCR assay, Western blot and immunohistochemistry

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Asian Journal of Pharmaceutical Sciences 2019年第3期14卷 329-339页

作者： Pan Guo Nan Li Lili Fan Jun Lu Boying Liu Bing Zhang Yumei Wu Zhidong Liu Jiawei Li Jiaxin Pi Dongli Qi Tianjin State Key Laboratory of Modern Chinese Medicine The Institute of Traditional Chinese MedicineTianjinUniversity of Traditional Chinese MedicineNo.88Yuquan RoadTianjin300193China Engineering Research Center of Modern Chinese Medicine Discovery and Preparation Tianjin University ofTraditional Chinese MedicineNo.88Yuquan RoadTianjin300193China School of Pharmaceutical Science and Technology Tianjin UniversityTianjin300193China

Labrasol, as a non-ionic surfactant, can enhance the permeation and absorption of drugs, and is extensively used in topical, transdermal, and oral pharmaceutical preparations as an emulsifier and absorption enhancer. Recent studies in our laboratory have indicated that labrasol has a strong absorption enhancing effect on different types of drugs in vitro and in vivo. This study was performed to further elucidate the action mechanism of labrasol on the corneal penetration. In this research, the fluorescein sodium, a marker of passive paracellular transport of tight junction, was selected as the model drug to assess the effect of labrasol on in vitro corneal permeability. To investigate the continuous and real-time influence of labrasol on the membrane permeability and integrity, the Ussing chamber system was applied to monitor the electrophysiological parameters. And, furthermore, we elucidated the effect of labrasol on excised cornea at the molecular level by application of RT-PCR, Western blot, and immunohistochemical staining. The results indicated that labrasol obviously enhance the transcorneal permeability of fluorescein sodium, and the enhancement was realized by interacting with and down-regulating the associated proteins, such as Factin, claudin-1 and β-catenin, which were contributed to cell-cell connections, respectively.

关键词： Enhanced corneal permeation Tight junctions associated proteins Labrasol Ussing chamber Western blot Immunohistochemistry

Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

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Science China(Life Sciences) 2009年第8期52卷 754-760页

作者： ZHAO Li1,HOU XinSheng5,JI Rong2,HAN ChunHui2,YU XiuPing3,HONG Tao4,5 1 Department of Laboratory Science of Microbiology,School of Public Health,Shandong University,Jinan 250012,China 2 Institute of Nutrition and Food Safety,Chinese Center of Disease Prevention and Control,Beijing 100021,China 3 Department of Microbiology,School of Medicine,Shandong University,Jinan 250012,China 4 Institute of Virus Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Beijing 100052,China 5 Institute of Biological Science and Technology,School of Science,Beijing Jiaotong University,Beijing 100044,China Department of Laboratory Science of Microbiology School of Public Health Shandong University Jinan China Institute of Biological Science and Technology School of Science Beijing Jiaotong University Beijing China Institute of Nutrition and Food Safety Chinese Center of Disease Prevention and Control Beijing China Department of Microbiology School of Medicine Shandong University Jinan China Institute of Virus Disease Prevention and Control Chinese Center for Disease Control and Prevention Beijing China

To obtain high titer monoclonal antibodies(McAbs) which can react with mammalian prion protein(PrP),Balb/C mice were immunized with bovine(Bo) PrP peptide(BoPrP 209-228 aa) coupled to keyhole limpet hemocyanin(KLH).The hybridoma cell lines secreting monoclonal antibodies against the peptide were established by cell fusion and cloning.The obtained McAbs were applied to detect recombinant human,bovine and hamster PrP,cellular prion protein(PrPc) in normal bovine brain and pathogenic scrapie prion protein(PrPSc) accumulated in the medulla oblongata of bovine spongiform encephalopathy(BSE)specimen with Western blot and immunohistochemical detection,respectively.The current procedure might offer a simple,feasible method to raise high titer antibodies for studying biological features of PrP in mammals,as well as detection of transmissible spongiform encephalopathy(TSE) and diagnosis of BSE,in particular.

关键词： prion monoclonal antibody Western blot immunohistochemistry ELISA

VARIATIONS OF LECTIN-BINDING GLYCOPROTEINS IN EARLY PREGNANT RABBIT EMBRYO

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Science China Chemistry 1992年第5期35卷 577-584页

作者：陈素珍曹咏清 Department of Endocrinology and Laboratory of Reproductive Biology Institute of Zoology Academia Sinica Beijing 100080 PRC

In the present study, the quantitative and qualitative changes of three kinds of lectinbinding glycoproteins of early pregnant rabbit embryos (D4—D12) were analyzed. The technique of Western blot, as well as video densitometer scanning and its analytic software was used for analysis. Results found that there were five specific lectin-binding glycoproteins in Day 4 to Day 6 blastocyst fluids: one ConA-binding glycoprotein about 70 kD, two WGA-binding glycoproteins respectively about 42 kD and 25kD and two PNA-binding glycoprotein respectively about 180kD and 75kD. They disappeared immediately after implan tation, It is demonstrated that there are stage-specific glycoproteins in rabbit blastocyst fluid which might be relevant to the recognition of pregnancy and implantation.

关键词： blastocyst fluid lectin glycoprotein Western blot implantation.

Molecular, serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

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Journal of Integrative Agriculture 2019年第11期18卷 2549-2560页

作者： LI Liu ZHENG Meng-meng MA Xiao-fang LI Yuan-jun LI Qing-yu WANG Guo-ping HONG Ni State Key Laboratory of Agromicrobiology Huazhong Agricultural UniversityWuhan 430070P.R.China Key Laboratory of Crop Disease Monitoring and Safety Control in Hubei Province College of Plant Science and TechnologyHuazhong Agricultural UniversityWuhan 430070P.R.China Yantai Academy of Agricultural Science Yantai 264000P.R.China

Apple stem pitting virus(ASPV)is an important causal agent of pear diseases.Nowadays,the infection status and molecular characteristics of the virus in old pear trees have never been investigated.In this study,we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar‘Chili’specifically grown at Laiyang area in China.ASPV-LYC possesses a chimeric genome consisting of 9273 nucleotides excluding a poly(A)tail at its 3′end and harboring a recombination region in its open reading frame(ORF1)with Aurora-1 and KL9 identified as the major and minor parents.Western blot analysis with antisera against recombinant coat proteins(CPs)of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates,but with differential activities.Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates.The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.

关键词： pear apple stem pitting virus genome Western blot