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24 ARTICLES PUBLISHED IN JoVE

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Biology

Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III
Luigi Boni 1, Gianlorenzo Dionigi 2, Francesca Rovera 1, Matteo Di Giuseppe 1
1Minimally Invasive Surgery Resarch Center, Department of Surgical Sciences, University of Insubria, 2Endocrine Surgery Research Center, University of Insubria

This video presents a case of laparoscopic left liver sectoriectomy perfomed in a 53 year old man suffering from Caroli's disease limited to segment II and III of the liver.

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Biology

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
Matteo Fossati 1,2,3, Nica Borgese 2,3,4, Sara Francesca Colombo 2,3, Maura Francolini 1,2
1Fondazione Filarete, 2Department of Biotechnology and Translational Medicine, University of Milan, 3Neuroscience Institute, National Research Council (CNR), 4Department of Health Science, "Magna Graecia" University of Catanzaro

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

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Medicine

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR
Marco Pacifici 1, Serena Delbue 2, Ferdous Kadri 1, Francesca Peruzzi 1
1Medical School and Stanley S. Scott Cancer Center, LSU Health Sciences Center, 2Department of Biomedical, Surgery and Dental Sciences, University of Milan

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

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Medicine

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury
Stephana Carelli *1, Toniella Giallongo *1, Claudio Gerace 1, Anthea De Angelis 1, Michele D. Basso 2, Anna Maria Di Giulio 1, Alfredo Gorio 1
1Laboratory of Pharmacology, Department of Health Sciences, University of Milan, 2Department of Neuroscience, College of Medicine, The Ohio State University

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

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Medicine

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
Wendy E. Heywood *1, Anna Baud *1, Emily Bliss 1, Ernestas Sirka 1, Jonathan M. Schott 2, Henrik Zetterberg 3, Daniela Galimberti 4, Neil J. Sebire 5, Kevin Mills 1
1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

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JoVE Journal

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes
Federica Franciosi 1, Irene Tessaro 1,2, Rozenn Dalbies-Tran 3, Cecile Douet 3, Fabrice Reigner 4, Stefan Deleuze 5, Pascal Papillier 3, Ileana Miclea 6, Valentina Lodde 1, Alberto M. Luciano 1, Ghylene Goudet 3
1Department of Health, Animal Science and Food Safety, University of Milan, 2IRCCS. Istituto Ortopedico Galeazzi, 3PRC, CNRS, IFCE, Université de Tours, INRA, 4PAO, INRA, 5Clinique des Animaux de Compagnie et des Équidés, Université de Liège, 6University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

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JoVE Core

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos
Lorena Benedetti 1,2,3, Anna Ghilardi 4, Laura Prosperi 4, Maura Francolini *1, Luca Del Giacco *4
1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 2Department of Neuroscience; Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, 4Department of BioSciences, Università degli Studi di Milano

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

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Biochemistry

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve
Cristina Banfi 1, Anna Guarino 2, Maura Brioschi 1, Stefania Ghilardi 1, Valeria Mastrullo 1, Elena Tremoli 1, Gianluca Polvani 2,3,4
1Centro Cardiologico Monzino IRCCS, 2Cardiovascular Tissue Bank of Milan, Centro Cardiologico Monzino IRCCS, 3Department of Clinical Sciences and Community Health, Cardiovascular Section, University of Milan, 4Department of Cardiovascular Disease, Development and Innovation Cardiac Surgery Unit, Centro Cardiologico Monzino IRCCS

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

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Immunology and Infection

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers
Giulia Ercoli 1, Gianluca Lopez 1,2, Camilla Ciapponi 1,3, Chiara Corti 1,2, Luca Despini 4, Donatella Gambini 5, Letterio Runza 1, Concetta Blundo 4, Amedeo Sciarra 1, Nicola Fusco 1,6
1Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 2School of Medicine, University of Milan, 3School of Biology, University of Pavia, 4Division of Breast Surgery, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, 5Division of Medical Oncology, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, 6Department of Biomedical, Surgical, and Dental Sciences, University of Milan

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

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Medicine

Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model
Che-Wei Wu 1,2,3, Tzu-Yen Huang 2, Hui-Chun Chen 4, Hsiu-Ya Chen 5, Tsung-Yi Tsai 2, Pi-Ying Chang 5, Yi-Chu Lin 2, Hsin-Yi Tseng 2, Pao-Chu Hun 6, Xiaoli Liu 7, Hui Sun 7, Gregory W. Randolph 8, Gianlorenzo Dionigi 9, Feng-Yu Chiang 2,3, I-Cheng Lu 5,10,11
1Department of Otorhinolaryngology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, 2Department of Otorhinolaryngology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 3Department of Otorhinolaryngology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, 4Department of Nursing, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 5Department of Anesthesiology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 6Laboratory Animal Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 7Department of Thyroid and Parathyroid Surgery, China-Japan Union Hospital and Jilin Provincial Key Laboratory of Surgical Translational Medicine, Jilin University, 8Division of Thyroid and Parathyroid Endocrine Surgery, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary; Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital; Department of Otology and Laryngology, Harvard Medical School, 9Division for Endocrine Surgery, Department of Human Pathology in Adulthood and Child-hood "G. Barresi", University Hospital G. Martino, University of Messina, 10Department of Anesthesiology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, 11Department of Anesthesiology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

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Immunology and Infection

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses
Fulvia Milena Cribiù *1, Claudia Burrello *2, Roberta Tacchi 1, Francesca Boggio 1, Dario Ricca 1, Flavio Caprioli 3,4, Stefano Ferrero 1, Federica Facciotti 2
1Pathology Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, 2Department of Experimental Oncology, European Institute of Oncology, 3Gastroenterology and Endoscopy Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, 4Department of Pathophysiology and Transplantation, Università degli Studi di Milano

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding human samples.

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Immunology and Infection

Isolation, Transfection, and Culture of Primary Human Monocytes
Karlie Plaisance-Bonstaff 1,2, Celeste Faia 2, Dorota Wyczechowska 1, Duane Jeansonne 1, Cecilia Vittori 1,3, Francesca Peruzzi 1,2
1Department of Medicine, Louisiana State University Health Sciences Center, 2Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, 3Department of Biomedical and Clinical Sciences L. Sacco, University of Milan

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

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Medicine

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis
Marco Invernizzi 1, Letterio Runza 2, Alessandro De Sire 1, Lorenzo Lippi 1, Concetta Blundo 3, Donatella Gambini 4, Renzo Boldorini 5,6, Stefano Ferrero 2,7, Nicola Fusco 2,7
1Physical and Rehabilitative Medicine, Department of Health Sciences, University of Eastern Piedmont "A. Avogadro", 2Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 3Division of Breast Surgery, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 4Division of Medical Oncology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 5Division of Pathology, Maggiore della Carità Hospital, 6Department of Health Sciences, University of Eastern Piedmont "A. Avogadro", 7Department of Biomedical, Surgical, and Dental Sciences, University of Milan

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

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Developmental Biology

Quantification of Self-renewal in Murine Mammosphere Cultures
Thalia Vlachou 1, Xieraili Aobuli 1, Errico D'Elia 1, Angela Santoro 1, Maria Cristina Moroni 1, Pier Giuseppe Pelicci 1,2
1Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 2Department of Oncology and Hemato-Oncology, University of Milan

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

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Neuroscience

Brain Morphology of Cannabis Users With or Without Psychosis: A Pilot MRI Study
Giuseppe Delvecchio *1, Lucio Oldani *2, Gian Mario Mandolini 2, Alessandro Pigoni 3, Valentina Ciappolino 2, Giandomenico Schiena 2, Matteo Lazzaretti 2, Elisabetta Caletti 2, Viviana Barbieri 1, Claudia Cinnante 4, Fabio Triulzi 1,4, Paolo Brambilla 1,2
1Department of Pathophysiology and Transplantation, University of Milan, 2Department of Neurosciences and Mental Health, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, 3MoMiLab Research Unit, IMT School for Advanced Studies Lucca, 4Neuroradiology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

This is a 3T magnetic resonance imaging study aiming to investigate grey matter volume differences between cannabis-induced psychosis patients and non-psychotic chronic cannabis users.

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Developmental Biology

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle
Rodrigo G. Barros 1, Valentina Lodde 1, Federica Franciosi 1, Alberto M. Luciano 1
1Reproductive and Developmental Biology Laboratory, Department of Health, Animal Science and Food Safety, University of Milan

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.  

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Cancer Research

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma
Greta Donisi *1, Marialuisa Barbagallo *2, Giovanni Capretti 1,3, Gennaro Nappo 1,3, Panteleimon G. Takis 4,5,6, Alessandro Zerbi 1,3, Federica Marchesi 2,7, Nina Cortese 2
1Section of Pancreatic Surgery, Humanitas Clinical and Research Center-IRCCS, 2Department of Immunology and Inflammation, Humanitas Clinical and Research Center-IRCCS, 3Department of Biomedical Sciences, Humanitas University, 4Giotto Biotech S.R.L., 5Section of Bioanalytical Chemistry, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, 6National Phenome Centre, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, 7Department of Medical Biotechnology and Translational Medicine, University of Milan

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

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Behavior

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test
Noemi Sofia Rabitti 1, Marta Appiani 1, Camilla Cattaneo 1, Rebecca Ford 2, Monica Laureati 1
1Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, 2Division of Food, Nutrition, and Dietetics, School of Biosciences, University of Nottingham

This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test.

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Challenges and Solutions For Female Fertility Preservation in Mammals
Federica Franciosi 1
1Department of Health, Animal Science and Food Safety, University of Milan

Challenges and Solutions For Female Fertility Preservation in Mammals

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Medicine

Investigation of the Electrophysiological and Thermographic Safety Parameters of Surgical Energy Devices During Thyroid and Parathyroid Surgery in a Porcine Model
Hsin-Yi Tseng 1, Tzu-Yen Huang 1, Jia Joanna Wang 1,2, Yi-Chu Lin 1, I-Cheng Lu 3, Feng-Yu Chiang 4, Gianlorenzo Dionigi 5,6, Gregory W. Randolph 7, Che-Wei Wu 1,8
1Department of Otorhinolaryngology-Head and Neck Surgery, International Thyroid Surgery Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 2Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 3Department of Anesthesiology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 4Department of Otolaryngology-Head and Neck Surgery, E-Da Hospital, I-Shou University, 5Division of General Surgery, Endocrine Surgery Section, Istituto Auxologico Italiano (IRCCS), 6Department of Pathophysiology and Transplantation, Faculty of Medicine and Surgery, University of Milan, 7Division of Thyroid and Parathyroid Endocrine Surgery, Department of Otolaryngology-Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 8Center for Liquid Biopsy and Cohort Research, and Faculty of Medicine, College of Medicine, Kaohsiung Medical University

The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery.

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Medicine

Transthoracic Echocardiography to Assess Post-Resuscitation Left Ventricular Dysfunction After Acute Myocardial Infarction and Cardiac Arrest in Pigs
Daria De Giorgio 1, Davide Olivari 1, Francesca Fumagalli 1, Lidia Staszewsky 1, Giuseppe Ristagno 2,3
1Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 2University of Milan, 3Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Transthoracic echocardiography is the first-line diagnostic test for post-resuscitation left ventricular dysfunction and structural changes in a pig model of cardiac arrest.

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Cancer Research

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management
Giuseppina Bonizzi *1, Maria Capra *1, Cristina Cassi 1, Giulio Taliento 1, Oriana Pala 2, Elham Sajjadi 2,3, Konstantinos Venetis 2,3, Mariia Ivanova 2, Massimo Monturano 4, Giuseppe Renne 2, Lorenzo Zattoni 1,3, Elena Guerini-Rocco 2,3, Giuseppe Viale 2,3, Roberto Orecchia 5, Nicola Fusco 1,2,3
1Biobank for Translational and Digital Medicine Unit, IEO, European Institute of Oncology IRCCS, 2Division of Pathology, IEO, European Institute of Oncology IRCCS, 3Department of Oncology and Hemato-Oncology, University of Milan, 4Patient Safety & Risk Management Service, IEO, European Institute of Oncology IRCCS, 5Scientific Directorate, IEO, European Institute of Oncology IRCCS

Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples.

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JoVE Core

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells
Rafael Badain 1, Daniel S. C. Damineli 1,2, Maria Teresa Portes 3, José Feijó 4, Stefano Buratti 5, Giorgia Tortora 6, Hugo Neves de Oliveira 1, Roberto M. Cesar Jr 1
1Institute of Mathematics and Statistics, University of São Paulo, 2Center for Mathematics, Computing and Cognition, Federal University of ABC, 3Department of Botany, Institute of Biosciences, University of São Paulo, 4Cell Biology and Molecular Genetics Department, University of Maryland, 5Department of Biosciences, University of Milan, 6Department of Physics, Politecnico di Milano

Current methods for analyzing the intracellular dynamics of polarized single cells are often manual and lack standardization. This manuscript introduces a novel image analysis pipeline for automating midline extraction of single polarized cells and quantifying spatiotemporal behavior from time lapses in a user-friendly online interface.

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Genetics

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types
Valeria Di Gioia 1,2, Jan Zamporlini 1,3, Rebecca Vadalà 1,3, Elena Parmigiani 4, Beatrice Bodega 1,3, Federica Marasca 1
1Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM), 2SEMM, European School of Molecular Medicine, 3Department of Biosciences, University of Milan, 4T-One Therapeutics Srl

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

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