Massachusetts General Hospital, Harvard Medical School 21 articles published in JoVE Biology Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle Yan Bai1, Xingbin Ai1 1Division of Newborn Medicine, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu. Bioengineering A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies Victor Pozzo1,2,4, Golda Romano1,2,4, Marion Goutard1,2,4, Elise Lupon1,2,4, Pierre Tawa1,2,4, Aylin Acun3,4,5, Alec R. Andrews2, Corentin B. Taveau1,2,4,6, Basak E. Uygun1,2,3,4, Mark A. Randolph1,2,4, Curtis L. Cetrulo1,2,4, Alexandre G. Lellouch1,2,4,6 1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, 4Shriners Hospital for Children, 5Department of Biomedical Engineering, Widener University, 6Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université Paris Descartes The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research. Medicine Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging Benjamin A. Kuzma1, Isaac J. Pence1, Alexander Ho1, Conor L. Evans1 1Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery. Immunology and Infection Partial Heterotopic Hindlimb Transplantation Model in Rats Marion Goutard1,2,3, Mark A. Randolph1,2,3, Corentin B. Taveau1,2,3,4, Elise Lupon1,2,3, Laurent Lantieri4, Korkut Uygun1,2,3, Curtis L. Cetrulo Jr.*1,2,3, Alexandre G. Lellouch*1,2,3,4 1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Shriners Hospital for Children, 4Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université de Paris This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up. Immunology and Infection Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus Hong-My Nguyen*1, Naresh Sah*1, Melissa R. M. Humphrey2, Samuel D. Rabkin2, Dipongkor Saha1 1Department of Immunotherapeutics and Biotechnology, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, 2Molecular Neurosurgery Laboratory and the Brain Tumor Research Center, Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use. Medicine A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects Riccardo Pinciroli1, Lisa Traeger1, Anna Fischbach1, Stefano Gianni1, Caio Cesar Araujo Morais1, Bijan Safaee Fakhr1, Raffaele Di Fenza1, Dianna Robinson3, Ryan Carroll2, Warren M. Zapol1, Lorenzo Berra1 1Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, 2Division of Pediatric Critical Care Medicine, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, 3Respiratory Care, Massachusetts General Hospital This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects. Neuroscience Real-Time fMRI Brain Mapping in Animals Sangcheon Choi1,2, Kengo Takahashi1,2, Yuanyuan Jiang1,3, Sascha Köhler4, Hang Zeng1,2, Qi Wang1,2, Yan Ma1,2, Xin Yu1,3 1Max Planck Institute for Biological Cybernetics, 2Graduate Training Centre of Neuroscience, 3MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Harvard Medical School, Massachusetts General Hospital, 4Bruker BioSpin Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI. Neuroscience Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits Mark P. Ottensmeyer1,2, Shasha Li2,3,4, Gianluca De Novi1,2, A. Aria Tzika2,3,4 1Medical Device & Simulation Laboratory, Department of Radiology, Massachusetts General Hospital, 2Harvard Medical School, 3NMR Surgical Laboratory, Department of Surgery, Center for Surgery, Innovation and Bioengineering, Massachusetts General Hospital, Harvard Medical School, 4Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits. Bioengineering Bulk Droplet Vitrification for Primary Hepatocyte Preservation Reinier J. de Vries1,2,3, Peony D. Banik1,2, Sonal Nagpal1,2, Lindong Weng1,2, Sinan Ozer1,2, Thomas M. van Gulik3, Mehmet Toner1,2, Shannon N. Tessier1,2, Korkut Uygun1,2 1Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, 2Shriners Hospitals for Children, Boston, 3Department of Surgery, University of Amsterdam This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets. Medicine Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model Che-Wei Wu1,2,3, Tzu-Yen Huang2, Hui-Chun Chen4, Hsiu-Ya Chen5, Tsung-Yi Tsai2, Pi-Ying Chang5, Yi-Chu Lin2, Hsin-Yi Tseng2, Pao-Chu Hun6, Xiaoli Liu7, Hui Sun7, Gregory W. Randolph8, Gianlorenzo Dionigi9, Feng-Yu Chiang2,3, I-Cheng Lu5,10,11 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. Developmental Biology Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells Shino Mitsunaga1, Keiko Shioda1, Kurt J. Isselbacher1, Jacob H. Hanna2, Toshi Shioda1 1Center for Cancer Research, Massachusetts General Hospital, 2Department of Molecular Genetics, Weizmann Institute of Science Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells. Immunology and Infection Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants Eliana Drenkard*1, Rhianna M. Hibbler*1, D. Alina Gutu2, Alexander D. Eaton1, Amy L. Silverio1, Frederick M. Ausubel2,3, Bryan P. Hurley1,4, Lael M. Yonker1,4 1Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, 2Department of Molecular Biology, Massachusetts General Hospital, 3Department of Genetics, Harvard Medical School, 4Department of Pediatrics, Harvard Medical School Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library. Neuroscience Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo Hana Uhlirova1,2, Peifang Tian3,4, Kıvılcım Kılıç3,5, Martin Thunemann1, Vishnu B. Sridhar6, Radim Chmelik2,7, Hauke Bartsch1, Anders M. Dale1,3, Anna Devor1,3,8, Payam A. Saisan3 1Department of Radiology, University of California, San Diego, 2Central European Institute of Technology, Brno University of Technology, 3Department of Neurosciences, University of California, San Diego, 4Department of Physics, John Carroll University, 5Department of Biomedical Engineering, Boston University, 6Bioengineering Undergraduate Program, University of California, San Diego, 7Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, 8Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data. Immunology and Infection In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging Yucheng Wang1,2,3, Olivia D. Harrington1, Ying Wang1, Clinton K. Murray4, Michael R. Hamblin1, Tianhong Dai1 1Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, 2Department of Medical Oncology, Beijing Institute of Translational Medicine, Chinese Academy of Sciences, 3Cancer Center, Aviation General Hospital, Beijing, 4Infectious Disease Service, Brooke Army Medical Center Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging. Biology Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM) Jing Men1,2, Jason Jerwick2,3, Penghe Wu1,2, Mingming Chen3,4, Aneesh Alex2,3, Yutao Ma4, Rudolph E. Tanzi5, Airong Li5, Chao Zhou1,2,3 1Bioengineering Program, Lehigh University, 2Center for Photonics and Nanoelectronics, Lehigh University, 3Department of Electrical and Computer Engineering, Lehigh University, 4State Key Laboratory of Software Engineering, Wuhan University, 5Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images. Medicine Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy Christos Papadelis1, Eleonora Tamilia1, Steven Stufflebeam2, Patricia E. Grant1, Joseph R. Madsen3, Phillip L. Pearl4, Naoaki Tanaka2 1 High Frequency Oscillations (HFOs) have emerged as presurgical biomarkers for the identification of the epileptogenic zone in pediatric patients with medically refractory epilepsy. A methodology for the noninvasive recording, detection, and localization of HFOs with simultaneous scalp electroencephalography (EEG) and magnetoencephalography (MEG) is presented. Bioengineering TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application Keumyeon Kim1, Haeshin Lee1,2, Seonki Hong2,3 1The Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), 2Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 3Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue. Bioengineering High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis Kim V. Berghaus1, Seok H. Yun2,3, Giuliano Scarcelli1 1Fischell Department of Bioengineering, University of Maryland, 2Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 3The Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo. Bioengineering Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization William J. McCarty1,2, Ljupcho Prodanov1,2, Shyam Sundhar Bale1,2, Abhinav Bhushan1,2, Rohit Jindal1,2, Martin L. Yarmush1,2, O. Berk Usta1,2 1Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, 2Shriners Hospitals for Children-Boston The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models. Medicine Ex Situ Normothermic Machine Perfusion of Donor Livers Negin Karimian1,2, Alix P.M. Matton1,2, Andrie C. Westerkamp1,2, Laura C. Burlage1,2, Sanna op den Dries1,2, Henri G.D. Leuvenink2, Ton Lisman1,2, Korkut Uygun3, James F. Markmann4, Robert J. Porte1,2 1Section of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, 2Surgical Research Laboratory, Department of Surgery, University of Groningen, University Medical Center Groningen, 3Center of Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 4Division of Transplantation, Department of Surgery, Massachusetts General Hospital, Harvard Medical School Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft. Medicine Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion Bote G. Bruinsma*1, James H. Avruch*2, Pepijn D. Weeder1, Gautham V. Sridharan1, Basak E. Uygun1, Negin G. Karimian1, Robert J. Porte3, James F. Markmann2, Heidi Yeh2, Korkut Uygun1 1Center for Engineering in Medicine, Dept. of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Transplant Center, Dept. of Surgery, Massachusetts General Hospital, Harvard Medical School, 3Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University Medical Center Groningen, University of Groningen We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 °C).
