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Third Military Medical University

20 ARTICLES PUBLISHED IN JoVE

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Neuroscience

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
Zhongshu Tang 1, Shuihua Zhang 1,2, Chunsik Lee 1, Anil Kumar 1, Pachiappan Arjunan 1, Yang Li 1, Fan Zhang 1, Xuri Li 1
1National Eye Institute, NIH, 2Ophthalmology Department, The Second Hospital of Harbin Medical University

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

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Medicine

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study
Zhongshu Tang 1, Fan Zhang 1, Yang Li 1, Pachiappan Arjunan 1, Anil Kumar 1, Chunsik Lee 1, Xuri Li 1
1National Eye Institute

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

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Bioengineering

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides
Yang Li 1, Catherine A. Foss 2, Martin G. Pomper 2,3, S. Michael Yu 1,3
1Department of Bioengineering, University of Utah, 2Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 3Institute for NanoBiotechnology, Johns Hopkins University

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

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Immunology and Infection

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells
Chun Yang *1, Wei Xiong *2, Qian Qiu 2, Houda Tahiri 1, Carmen Gagnon 1, Guoxiang Liu 2, Pierre Hardy 1
1Departments of Pediatrics and Pharmacology, University of Montréal, 2Department of Pulmonology, Chongqing Southwest Hospital, Third Military Medical University

Cell membrane–shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

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Biochemistry

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells
Xiongwei Cai 1,2,3, Yi Zheng 1, Nancy A. Speck 2
1Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, 2Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, 3Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

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Cancer Research

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation
Juan Wang 1, Jin Wen 2, Xiao-Xiang Chen 1, Guang-Liang Chen 3
1Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 2Department of Nephrology and Rheumatology, Yongchuan Hospital of Chongqing Medical University, 3Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

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Behavior

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats
Lupeng Yue 1,2, Fengrui Zhang 3, Xuejing Lu 1,2, You Wan 4,5,6, Li Hu 1,2,7
1CAS Key Laboratory of Mental Health, Institute of Psychology, 2Department of Psychology, University of Chinese Academy of Sciences, 3Research Center of Brain Cognitive Neuroscience, Liaoning Normal University, 4Neuroscience Research Institute, Peking University, 5Department of Neurobiology, School of Basic Medical Sciences, Peking University, 6Key Laboratory for Neuroscience, Ministry of Education/National Health and Family Planning Commission, Peking University, 7Department of Pain Management, State Key Clinical Specialty in Pain Medicine, Second Affiliated Hospital of Guangzhou Medical University

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

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JoVE Core

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea
Shu-Kui Yu *1, Zheng-De Du *1, Qing-Ling Song 1, Teng-Fei Qu 1, Yue Qi 1, Wei Xiong 1, Lu He 1, Wei Wei 2, Shu-Sheng Gong 1, Ke Liu 1
1Department of Otolaryngology-Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, 2Department of Otology, Shengjing Hospital of China Medical University

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

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Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues
Chang Liu 1, Shuangshuang Wang 1, Man Liu 2, Fuxiang Bai 1, Zhihong Chen 3, Ping Wang 4, Jun Mi 1,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Shijiazhuang Shimen Experimental School, 3Qilu Children's Hospital of Shandong University, 4Qilu Hospital of Shandong University, 5Shenzhen Research Institute of Shandong University

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

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Cancer Research

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
Lisha Wang 1, Zhiming Wang 1, Junyi Guo 2, Huayu Lin 1, Shuqiong Wen 2, Qiao Liu 1, Yiding Li 3, Qing Wu 1, Leiqiong Gao 1, Xiangyu Chen 1, Luoyingzi Xie 1, Qin Tian 1, Jianfang Tang 1, Zhirong Li 1, Li Hu 1, Juan Wang 4, Lifan Xu 1, Qizhao Huang 5, Lilin Ye 1
1Institute of Immunology, Third Military Medical University, 2Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Stomatological Hospital, Sun Yat-Sen University, 3Shigatse Branch, Xinqiao Hospital, Third Military Medical University, 4Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, 5Cancer Center, The General Hospital of Western Theater Command

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

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Biochemistry

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay
Lisha Wang 1, Liza Bergkvist 1, Rajnish Kumar 1,2, Bengt Winblad 1,3, Pavel F. Pavlov 1
1Department of Neuroscience Care and Society, Division of Neurogeriatrics, Karolinska Institutet, 2Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (BHU), 3Theme Inflammation and Aging, Karolinska University Hospital

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

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Bioengineering

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632
Zhiwei Xie 1,2,3, Jizhou Shi 4, Min Zong 3, Qiuping Xu 1, Chang Liu 1, Jie Wen 1, Qun Zhang 1, Panpan Liu 1, Guanyi Liu 1, Jing Guo 2,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 3Department of Stomatology, Shengli Oilfield Central Hospital, 4Department of Pediatric Surgery, Shengli Oilfield Central Hospital, 5Ningbo Stomatology Hospital of Savaid Stomatology School, Hangzhou Medical College

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

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Neuroscience

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System
Xiaorui Wang 1, Yixun Su 1,2, Xuelian Hu 1,3, Jianqin Niu 1
1Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Brain, and Intelligence Research Key Laboratory of Chongqing Education Commission, Third Military Medical University, 2Research Centre, The Seventh Affiliated Hospital of Sun Yat-sen University, 3School of Medicine, Chongqing University

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

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Medicine

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method
Xiaoli Jin *1, Yang Li *2, Xuanwei Chen 1, Jin Chen 1, Jian Xu 1
1School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, 2School of Basic Medical Sciences, Fudan University

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

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Bioengineering

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain
Rang Feng 1, Guo-Cheng Li 1, Hai-Ming Jing 1, Chang Liu 1, Ruo-Yi Xue 1, Quan-Ming Zou 1, Hai-Bo Li 1
1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

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Immunology and Infection

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles
Qin Xiao *1, Yuheng Liu *1, Dandan Zhang 1, Chao Li 1, Qihua Yang 1, Dongshui Lu 1, Weijun Zhang 1, Joseph Rosenecker 2, Quanming Zou 1, Yang Li 3, Shan Guan 1
1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, Third Military Medical University, 2Department of Pediatrics, Ludwig-Maximilians University of Munich, 3Department of Pharmacy, Southwest Hospital, Third Military Medical University

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

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Immunology and Infection

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection
Xiaoqiang Zeng 1, Hongwu Sun 1, Yan Ye 1, Xing Luo 1, Dingyi Cai 1, Yun Yang 1, Ting Chen 1, Cun Sun 1, Shaotong Zhang 1, Hao Zeng 1
1National Engineering Research Centre of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

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Immunology and Infection

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis
Yan Zhang *1, Xingxing Su *2, Lisha Wang 2, Zhengliang Yue 2, Qiao Liu 3, Ling Ran 2, Shun Lei 2, Jianjun Hu 2, Lifan Xu 2, Lilin Ye 2, Ping Ji 4, Guimei Li 1, Qizhao Huang 5, Shuqiong Wen 4
1College of Veterinary Medicine, Qingdao Agricultural University, 2Institute of Immunology, Third Military Medical University, 3Institute of Cancer, Xinqiao Hospital, Third Military Medical University, 4Stomatological Hospital of Chongqing Medical University, 5Institute of life science, Chongqing Medical University

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

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Immunology and Infection

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses
Guo-Cheng Li 1, Hui-Fang Li 1, Zhe Jin 1, Rang Feng 1, Yan Deng 1, Hao Cheng 1, Hai-Bo Li 1
1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

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Immunology and Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice
Yao Lin *1,2, Shuai Yue *3, Yang Yang *4, Junjian He 5, Xiaofan Yang 6, Lilin Ye 5, Xiangyu Chen 7
1Department of Urology, South China Hospital, Medical School, Shenzhen University, 2Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, 3Cancer Center, Daping Hospital & Army Medical Center of PLA, Third Military Medical University, 4Guangdong Provincial Key Laboratory of Immune Regulation and Immunotherapy, School of Laboratory Medicine and Biotechnology, Southern Medical University, 5Institute of Immunology, Third Military Medical University, 6Dermatology Hospital, Southern Medical University, 7Institute of Immunological Innovation and Translation, Chongqing Medical University

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

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