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Peking University Third Hospital

20 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue
F. Javier Rubio *1, Xuan Li *1, Qing-Rong Liu 1, Raffaello Cimbro 2, Bruce T. Hope 1
1Behavioral Neuroscience Research Branch, Intramural Research Program, NIDA, NIH, DHHS, 2Division of Rheumatology, School of Medicine, Johns Hopkins University

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

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Neuroscience

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization
Xiao Xu 1, Yinwei You 1,2, Long Zhang 1
1Department of Entomology, China Agricultural University, 2Bio-tech Research Center, Shandong Academy of Agricultural Sciences

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

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Bioengineering

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex
Rong Li *1, Jinxin Miao *1,2, Zhiqiang Fan 1, SeokHwan Song 3, Il-keun Kong 3,4, Yaohe Wang 2,5, Zhongde Wang 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2National Centre for International Research in Cell and Gene Therapy, Sino-British Research Centre, School of Basic Medical Sciences, Zhengzhou University, 3Department of Animal Science Division of Applied Life Science (BK21 Plus), Gyeongsang National University, 4Institute of Agriculture and Life Science, Gyeongsang National University, 5Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

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Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers
Huijuan You *1, Shimin Le *2,3, Hu Chen 2,4, Linyan Qin 1, Jie Yan 2,4,5
1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 2Mechanobiology Institute, National University of Singapore, 3Department of Physics, National University of Singapore, 4Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, 5Center for Bioimaging Sciences, National University of Singapore

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

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Cancer Research

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Xiaoxing Lv 1, Meiru Zhao 1, Yuting Yi 1, Lucheng Zhang 1, Yanfang Guan 1, Tao Liu 1, Ling Yang 1, Rongrong Chen 1, Jianhui Ma 1, Xin Yi 1
1Geneplus-Beijing Institute

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

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Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array
Jianwei Chen *1,2,3, Ning Shao *1,2,3, Jiaying Hu *4, Rong Li 4, Yuanshou Zhu 1,2,3, Dabing Zhang 4,5, Shujuan Guo 1,2,3, Junhou Hui 6, Peng Liu 6, Litao Yang 4, Sheng-Ce Tao 1,2,3
1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 2State Key Laboratory of Oncogenes and Related Genes, 3School of Biomedical Engineering, Shanghai Jiao Tong University, 4Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 5Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, 6Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

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Neuroscience

Single Sensillum Recordings for Locust Palp Sensilla Basiconica
Hongwei Li 1, Yinwei You 2, Long Zhang 1
1Department of Entomology, China Agricultural University, 2Bio-tech Research Center, Shandong Academy of Agricultural Sciences

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

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Neuroscience

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons
Mengye Zhu 1, Daying Zhang 1, Sicong Peng 2, Nana Liu 2, Jing Wu 2, Haixia Kuang 2, Tao Liu 2,3
1Department of Pain, First Affiliated Hospital of Nanchang University, 2Department of Pediatrics, First Affiliated Hospital of Nanchang University, 3Center for Laboratory Medicine, First Affiliated Hospital of Nanchang University

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

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Medicine

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy
Wen-Hui Wang 1, Long Zhang 2,3, Guo-Xiang Dong 2, Jun Zhao 2, Xuan Li 2,3
1Department of Dermatology, Peking University Third Hospital, 2Department of Interventional Radiology and Vascular Surgery, Peking University Third Hospital, 3Wound Healing Center, Peking University Third Hospital

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

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Cancer Research

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma
Yu Zhang 1, Yupeng Yao 1, Yan Zhang 1
1MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

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Neuroscience

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential
Xiang Fan 1,2, Lingling Wu 1,2, Aihua Ding 1,2
1Department of Ophthalmology, Peking University Third Hospital, 2Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

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Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery
Tao Liu *1,2,3,4,5, Xueling Song *1,2,3,4,5, Xiaoying Zheng *1,2,3,4,5, Liying Yan 1,2,3,4,5, Caihong Ma 1,2,3,5, Rong Li 1,2,3,4,5, Jie Yan 1,2,3,4,5, Jie Qiao 1,2,3,4,5
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology, 3Key Laboratory of Assisted Reproduction, Ministry of Education, 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, 5Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

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Neuroscience

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues
Faraz Ahmad 1, Ping Liu 1
1Department of Anatomy, School of Biomedical Sciences, University of Otago

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

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Genetics

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis
Jin Huang *1,2,3,4, Yaxin Yao *5, Jialin Jia 1,2,3,4, Xiaohui Zhu 1,2,3,4, Jieliang Ma 5, Jing Wang 5, Ping Liu 1,2,3,4, Sijia Lu 5
1Centre for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology(Peking University Third Hospital), 3Ministry of Education, Key Laboratory of Assisted Reproduction (Peking University), 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, 5Department of Clinical Research, Yikon Genomics Co. Ltd.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

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Medicine

Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model
Yuanmin Yang *1, Shuoqiu Deng *1, Shuiqing Qu 1, Yu Zhang 1, Zhongyuan Zheng 1, Lina Chen 1, Yujie Li 1
1China Academy of Chinese Medical Sciences

This protocol describes a composite animal model with exposure to particulate matter (PM) that aggravates myocardial ischemia with atherosclerosis.

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Medicine

Iris Fixation via External Pentagram Suturing
Xiang Fan 1,2, Yansheng Hao 1,3, Ying Chu 1,2, Jing Hong 1,2
1Department of Ophthalmology, Peking University Third Hospital, 2Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, 3Xian Aier Eye Hospital

The present protocol describes how a 10-0 polypropylene suture crosses the anterior surface of the iris, forming a pentagram to prevent both iris and pupil from moving toward the cornea. This can be combined with subsequent keratoplasty to cure bullous keratopathy with extensive anterior synechia of the iris.

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Biology

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse
Jian Wu *1, Shijun Wang *1, Le Kang *1, Chunjie Yang *1, Jianguo Jia 1, Xingxu Wang 2, Ge Wei 1, Xuan Li 1, Jieyun You 2, Yunzeng Zou 1
1Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, 2Department of Cardiovascular Medicine, Shanghai East Hospital, School of Medicine, Tongji University

This protocol presents a practical guide on the surgery for creation of aortic regurgitation (AR) in the mouse. Assessment of the AR mouse by echocardiography and invasive hemodynamic measurement recapitulates its clinically relevant characteristics of volume overload-induced eccentric hypertrophy, suggesting its promising application in the study of cardiac hypertrophy.

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Medicine

Binocular Dynamic Visual Acuity in Eyeglass-Corrected Myopic Patients
Yuexin Wang *1, Yining Guo *1, Shanshan Wei 2, Yifei Yuan 1, Tingyi Wu 1, Yu Zhang 1, Yueguo Chen 1, Xuemin Li 1
1Department of Ophthalmology, Beijing Key Laboratory of Restoration of Damaged Ocular Nerves, Peking University Third Hospital, 2Beijing Tongren Eye Center, Beijing Tongren Hospital

The present research demonstrates a method to accurately examine dynamic visual acuity (DVA) in myopic subjects with eyeglass correction. Further analysis indicated that the closer the refraction state to emmetropia, the better the eyeglass-corrected binocular DVA is at both 40 and 80 degrees per second.

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Biology

Therapeutic Effects of Huazhuojiedu Decoction on Precancerous Lesions of Gastric Cancer by Regulating Mitophagy
Tianxiao Yang 1,2, Pingping Zhou 2, Ziwei Zhang 1, Xinyu Hao 3, Shaowei Liu 1,2, Haoyu Chen 1,2, Yuhua Wang 1,2, Yangang Wang 1,4
1Hebei University of Chinese Medicine, 2The First Affiliated Hospital of Hebei University of Chinese Medicine, 3Peking University Third Hospital, 4Beijing University of Chinese Medicine Third Affiliated Hospital

Here, we introduce a protocol that demonstrates the therapeutic efficacy of Huazhuojiedu decoction (HZJD) in alleviating precancerous lesions of gastric cancer using mitophagy regulation.

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Medicine

Rotating the Intraocular Lens to Prevent Posterior Capsular Opacification in Cataract Surgeries
Di Zhang *1,2, Ziyuan Liu *1,2, Hongyuan Cai 1,2, Haikun Wang 1,2, Xiaoyong Chen 1,2, Chun Zhang 1,2
1Department of Ophthalmology, Peking University Third Hospital, 2Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital

The present protocol describes removing residual epithelial cells by rotating the intraocular lens in extracapsular cataract surgery without extra tools for preventing posterior capsular opacification.

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