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32 ARTICLES PUBLISHED IN JoVE

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Medicine

Programmed Electrical Stimulation in Mice
Na Li 1, Xander H.T Wehrens 1,2
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)

Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates.

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Engineering

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates
Anirban Chakraborty 1, Xinchuan Liu 1, Cheng Luo 1
1Mechanical and Aerospace Engineering, University of Texas at Arlington

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

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Medicine

Technical Aspects of the Mouse Aortocaval Fistula
Kota Yamamoto 1,2, Xin Li 1,3, Chang Shu 3, Tetsuro Miyata 2, Alan Dardik 1,4
1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

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Biology

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy
Takashi Sakurai 1,2, Anthony Lanahan 2, Melissa J. Woolls 2, Na Li 2, Daniela Tirziu 2, Masahiro Murakami 2
1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

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Biology

Isolation and Primary Culture of Mouse Aortic Endothelial Cells
Jie-Mei Wang 1, Alex F. Chen 2, Kezhong Zhang 1
1Center for Molecular Medicine and Genetics, Wayne State University, 2Third Xiangya Hospital and the Institute of Vascular Disease and Translational Medicine, Central South University

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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Medicine

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava
Hualong Bai 1,2,3,4,5, Xin Li 6, Takuya Hashimoto 1,2,5, Haidi Hu 1,2,5, Trenton R. Foster 1,2,5, Jesse J. Hanisch 1,2,5, Jeans M. Santana 1,2,5, Alan Dardik 1,2,5
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

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JoVE Core

Preparation and High-temperature Anti-adhesion Behavior of a Slippery Surface on Stainless Steel
Pengfei Zhang 1, Chen Huawei 1, Guang Liu 1, Liwen Zhang 1, Deyuan Zhang 1
1School of Mechanical Engineering and Automation, Beihang University

Slippery surfaces provide a new way to solve the adhesion problem. This protocol describes how to fabricate slippery surfaces at high temperatures. The results demonstrate that the slippery surfaces showed anti-wetting for liquids and a remarkable anti-adhesion effect on soft tissues at high temperatures.

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Cancer Research

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome
Xianquan Zhan 1,2,3,4, Yuda Huang 1,2,3, Ying Long 1,2,3
1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4The State Key Laboratory of Medical Genetics, Central South University

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

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Genetics

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Xuan Yao 1,2, Xing Wang 1,2, Junlai Liu 2,3,4, Linyu Shi 1, Pengyu Huang 3, Hui Yang 1
1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 2College of Life Sciences, University of Chinese Academy of Sciences, 3School of Life Science and Technology, Shanghai Tech University, 4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

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Engineering

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy
Tianzhuo Zhao 1,2,3, Xin Li 1,2, Qixiu Zhong 1,2, Hong Xiao 1,3, Shuzhen Nie 1,3, Fuqiang Lian 1,3, Sining Sun 4, Zhongwei Fan 1,2,3
1The Academy of Opto-electronics, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3National Engineering Research Center for DPSSL, 4Beijing GK Laser Technology Co., Ltd.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

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Biology

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay
Courtney M. Anderson 1, Annelies Laeremans 1, Xiao-Ming Mindy Wang 1, Xingyong Wu 1, Bingqing Zhang 1, Emerald Doolittle 1, Jeffrey Kim 1, Na Li 1, Helly Xiao Yan Pimentel 1, Emily Park 1, Xiao-Jun Ma 1
1Advanced Cell Diagnostics, Inc

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

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Cancer Research

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue
Guang Lei *1, Haiyan Yang *1, Ting Hong 2, Xiangyu Zhang 1, Nong Yang 1, Yongchang Zhang 1
1Department of Medical Oncology, Lung Cancer and Gastrointestinal Unit, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, 2Hunan ClinicaI Research Center in Gynecologic Cancer, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

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Medicine

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats
Hui Yang 1, Pu Zou 1, Jingyuan Chen 1, Gaoxing Shi 1, Chenlu Wu 1, Mi Wang 1, Qin Zhou 2, Shenghua Zhou 1
1Department of Cardiology, The Second Xiangya Hospital, Central South University, 2Department of General Surgery, The Second Xiangya Hospital, Central South University

Here, we present a protocol to collect blood samples from the rat subclavian vein.

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Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity
Mengrou Liu *1, Chunsen Zhu *1, Shun Bai *2, Xin Li 1, Kaiqiang Fu 1, Lan Ye 1, Ke Zheng 1
1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

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Immunology and Infection

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue
Xin Li 1, Zhengmei Mao 2, Li Yang 1, Kai Sun 1
1Center for Metabolic and Degenerative Diseases, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 2Microscopy Core, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

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Biochemistry

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity
Meng Zhao 1, Yawen Tang 1, Patrick J. Ernst 1,2, Asher Kahn-Krell 1, Chengming Fan 1,3, Danielle Pretorius 1, Hanxi Zhu 1, Xi Lou 1, Lufang Zhou 2, Jianyi Zhang 1, Wuqiang Zhu 1
1Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham, 2Division of Cardiovascular Diseases, School of Medicine, School of Engineering, University of Alabama at Birmingham, 3Department of Cardiac Surgery, The Second Xiangya Hospital of Central South University

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

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Medicine

Blood Collection Through Subclavian Vein Puncture in Mice
Hui Yang *1, Chenlu Wu *1, Fengjiao Liu 2, Mi Wang 1, Pu Zou 1, Yuhu He 1, Qiming Liu 1, Qin Zhou 3, Shenghua Zhou 1
1Department of Cardiology, The Second Xiangya Hospital, Central South University, 2Department of Cardiology, The Xiangya Hospital, Central South University, 3Department of General Surgery, The Second Xiangya Hospital, Central South University

Here, we present a protocol to take blood samples from the subclavian vein of mice.

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Medicine

A Cryoinjury Model to Study Myocardial Infarction in the Mouse
Dong Wang *1,2, Grigol Tediashvili *1,2, Xiaomeng Hu 1,2, Alessia Gravina 2, Sivan G. Marcus 1,2, Hao Zhang 4, Jeffrey E Olgin 4, Tobias Deuse 1,2,5, Sonja Schrepfer 1,2,3,5
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco, 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Division of Cardiology, Cardiovascular Research Institute, University of California San Francisco, 5Cardiovascular Surgery, University Heart Center

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

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Behavior

Assessment of Sexual Behavior of Male Mice
Zi-Wei Liu 1, Ning Jiang 2, Xue Tao 2, Xiao-Ping Wang 1, Xin-Min Liu 2, Shui-Yuan Xiao 3
1Department of Psychiatry, The Second Xiangya Hospital, Central South University, National Clinical Research Center for Mental Disorders and National Technology Institute on Mental Disorders, Hunan Key Laboratory of Psychiatry and Mental Health, 2Research Center for Pharmacology & Toxicology, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, 3Department of Social Medicine and Health Management, Xiangya School of Public Health, Central South University

This article describes how to perform sexual behavior tests in male mice.

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Cancer Research

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis
Xianquan Zhan 1,2,3,4, Huanni Li 5, Shehua Qian 1,2,3, Xiaohan Zhan 1,2,3, Na Li 1,2,3
1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 5Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

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Cancer Research

Analysis of Side Population in Solid Tumor Cell Lines
Xiaoli Dong 1, Yingying Wei 1, Tao Xu 1, Xiaoyue Tan 1,2,3, Na Li 1,2,3
1School of Medicine, Nankai University, 2Tianjin Key Laboratory of Tumour Microenvironment and Neurovascular Regulation, 3Collaborative Innovation Center for Biotherapy, Nankai University

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

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Immunology and Infection

Optimization of the Cuff Technique for Murine Heart Transplantation
Yunhan Ma *1,2, Baiyi Xie *1,2, Helong Dai *3,4,5, Chenxi Wang 6, Shujiao Liu 6, Tianshu Lan 7, Shuangyue Xu 1,2, Guoliang Yan 1,2,6, Zhongquan Qi 1,2,8
1Organ Transplantation Institute, School of Medicine, Xiamen University, 2Fujian Key Laboratory of Organ and Tissue Regeneration, 3Department of Kidney Transplantation, Center of Organ Transplantation, The Second Xiangya Hospital of Central South University, 4Clinical Research Center for Organ Transplantation in Hunan Province, 5Clinical Immunology Center, Central South University, 6School of Medicine, Xiamen University, 7Xiamen Medical College, 8Medical College, Guangxi University

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

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Biology

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes
Xinyi Liu 1,2, Si Wu 1,2, Wenqian Li 1,2, Meihong Zhang 1,2, Yan Wu 1,2, Ning Zhou 3, Pa Wu 1,2
1Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, College of Life Science, Hunan Normal University, 2State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory for Microbial Molecular Biology, College of Life Science, Hunan Normal University, 3Department of Infectious Diseases, the Second Xiangya Hospital, Central South University

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

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Immunology and Infection

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis
Wei Liao 1,2, Chaoyu Ma 1, Nu Zhang 1
1Department of Microbiology, Immunology and Molecular Genetics, School of Medicine, University of Texas Health Science Center at San Antonio, 2Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, The Second Xiangya Hospital, Central South University

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

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Biochemistry

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells
Kendall Martin 1, Tong Zhang 1, Pengfei Zhang 2, William B. Chrisler 1, Fillmore L. Thomas 3, Fen Liu 4, Tao Liu 1, Wei-Jun Qian 1, Richard D. Smith 1, Tujin Shi 1
1Biological Sciences Division, Pacific Northwest National Laboratory, 2NHC Key Laboratory of Cancer Proteomics, Department of Oncology, Xiangya Hospital, Central South University, 3Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 4Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

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Medicine

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter
Zhaokai Li *1,2, Liu Liu *1, Zhong Wang 1
1Department of Cardiac Surgery, Cardiovascular Center, The University of Michigan, Ann Arbor, 2Department of Cardiovascular Medicine, Xiangya Hospital, Central South University

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

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Immunology and Infection

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis
Li Yaozhen 1, Wu Kemin 1, Ji Xiaoyu 1, Ouyang Yang 1, Tang Hongying 1, Pan Baihong 1
1Department of Vascular Surgery, Xiangya Hospital, Central South University

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

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Medicine

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China
Cheng Luo *1, Chengming Fan *1, Hao Zhang 1, Long Song 1, Yuhong Liu 1, Liming Liu 1
1Department of Cardiovascular Surgery, the Second Xiangya Hospital, Central South University

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

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Medicine

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice
Xun Wu 1, Kele Qin 1, Di Wang 2, Kun Xiang 1, Jun Peng 2, Jianjun Guo 3, Jinfu Yang 1,2, Chengming Fan 1,2,3
1Department of Cardiovascular Surgery, Second Xiangya Hospital, Central South University, 2Hunan Provincial Key Laboratory of Cardiovascular Research, 3Hunan Fangsheng Pharmaceutical Co., Ltd.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

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Medicine

Repeated Transcranial Magnetic Stimulation Combined with Action Observation Training in Children with Spastic Cerebral Palsy
Taolin Fan 1, Huiqun Wei 1, Jin'e Dai 1, Gang You 2, Zhihui Lu 3
1Department of Children's Rehabilitation Treatment, Xiangya Boai Rehabilitation Hospital, 2Department of Rehabilitation, Xiangya Hospital, Central South University, 3The Public Course Teaching Department, Changsha Health Vocational College

Children with spastic cerebral palsy (SCP) have limb spasticity, movement disorders, and abnormal posture due to injury to the cerebral cortex motor area, resulting in inability to stand and walk normally. Therefore, alleviating limb spasticity and enhancing gross motor function in children with SCP have become important therapeutic goals.

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Medicine

Combination of Anatomical Plates and Rotator Cuff Reinforcement for Minimally Invasive Treatment of Greater Tuberosity Fractures of the Humerus
Xiao Lu *1, Xiaodi Zou *2, Ahmad Alhaskawi 3, Yanzhao Dong 3, Sohaib Hasan Abdullah Ezzi 4, Vishnu Goutham Kota 5, Mohamed Hasan Abdulla Hasan Abdulla 5, Sahar Ahmed Abdalbary 6, Hui Lu 3,7
1Department of Orthopedics, Sanmen People's Hospital, 2Department of Orthopedics, The Second Affiliated Hospital of Zhejiang Chinese Medical University, 3Department of Orthopedics, The First Affiliated Hospital, Zhejiang University, 4Department of Orthopedics, Third Xiangya Hospital, Central South University, 5Zhejiang University School of Medicine, 6Department of Orthopedic Physical Therapy, Faculty of Physical Therapy, Nahda University in Beni Suef, 7Alibaba-Zhejiang University Joint Research Center of Future Digital Healthcare, Zhejiang University

Here, we present a protocol to utilize anatomical plates alongside rotator cuff reinforcement in a minimally invasive surgery to efficiently treat humerus greater tuberosity fractures, promising quicker healing and improved stability.

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Medicine

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality
Maryam Moossavi *1,2, Hong Zhang *3,4, Jiarong Li 1,2,4, Feixiang Yan 1,2, Xiaolei Xu 1,2
1Department of Biochemistry and Molecular Biology, Mayo Clinic, 2Department of Cardiovascular Medicine, Mayo Clinic, 3Cardiovascular Surgery Department, Guilin Hospital of 2nd Xiangya Hospital, Central South University, 4Cardiovascular Surgery Department, 2nd Xiangya Hospital, Central South University

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

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