Pennsylvania State University College of Medicine 14 articles published in JoVE Biology Assessment of Bone Fracture Healing Using Micro-Computed Tomography Hwabok Wee*1,2, Deepak Kumar Khajuria*1,2, Fadia Kamal1,2,3, Gregory S. Lewis1,2, Reyad A. Elbarbary1,2,4 1Center for Orthopaedic Research and Translational Science (CORTS), The Pennsylvania State University College of Medicine, 2Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, 3Department of Pharmacology, The Pennsylvania State University College of Medicine, 4Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine Micro-computed tomography (µCT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on µCT procedures for analyzing the bone healing callus. This study provides a step-by-step µCT protocol that allows the monitoring of fracture healing. Biology Deep Learning-Based Segmentation of Cryo-Electron Tomograms Jessica E. Heebner1, Carson Purnell1, Ryan K. Hylton1, Mike Marsh2, Michael A. Grillo1, Matthew T. Swulius1 1Pennsylvania State University-College of Medicine, 2Object Research Systems This is a method for training a multi-slice U-Net for multi-class segmentation of cryo-electron tomograms using a portion of one tomogram as a training input. We describe how to infer this network to other tomograms and how to extract segmentations for further analyses, such as subtomogram averaging and filament tracing. Neuroscience Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin Mashanipalya G. Jagadeeshaprasad1, Prem Kumar Govindappa1, Amanda M. Nelson2, John C. Elfar1 1Department of Orthopaedics and Rehabilitation, Center for Orthopaedic Research and Translational Science (CORTS), The Pennsylvania State University College of Medicine, 2Department of Dermatology, The Pennsylvania State University College of Medicine The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin. Biology 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation Christina Li1, Jeel Shah1, Kylee Wrath1, Dahlia Matouba1, Connor Mills1, Kishore Punnath2, Ansu Perekatt1 1Department of Biology and Chemical Biology, Stevens Institute of Technology, 2Department of Pharmacology, Pennsylvania State University College of Medicine The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential. Medicine Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model William J. Pinamont1, Natalie K. Yoshioka1, Gregory M. Young1, Vengadeshprabhu Karuppagounder1, Elijah L. Carlson1, Adeel Ahmad1, Reyad Elbarbary1,2, Fadia Kamal1,3 1Center for Orthopedic Research and Translational Sciences, Department of Orthopedics and Rehabilitation, Pennsylvania State College of Medicine, 2Department of Biochemistry and Molecular Biology, Pennsylvania State College of Medicine, 3Department of Pharmacology, Pennsylvania State College of Medicine The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis. Genetics HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries Huacheng Luo1, Amin Sobh2, Christopher D. Vulpe2, Edmond Brewer1, Sinisa Dovat1, Yi Qiu3, Suming Huang1 1Department of Pediatrics, Pennsylvania State University College of Medicine, 2Department of Physiological Sciences, University of Florida, 3Department of Anatomy and Cell Biology, University of Florida A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci. Immunology and Infection An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis Shirley Albano-Aluquin1, Jozef Malysz2, Michal Kidacki3, Manohar Ratnam4, Nancy J. Olsen1 1Department of Medicine, Penn State University, 2Department of Pathology, Penn State University, 3Penn State College of Medicine, 4Department of Oncology, Wayne State University School of Medicine The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis. Immunology and Infection Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice Nathalie Fuentes1, Patricia Silveyra1,2 1Pulmonary, Immunology and Physiology Laboratory, Department of Pediatrics, Pennsylvania State University College of Medicine, 2Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle. Bioengineering Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography Alex Y. Lin1,2, Yifu Ding1,2,3, Daniel J. Vanselow1,2, Spencer R. Katz1,2,3, Maksim A. Yakovlev1,2, Darin P. Clark4, David Mandrell5, Jean E. Copper1,2, Damian B. van Rossum1,2, Keith C. Cheng1,2 1The Jake Gittlen Laboratories for Cancer Research, Penn State College of Medicine, 2Division of Experimental Pathology, Department of Pathology, Penn State College of Medicine, 3Medical Scientist Training Program, Penn State College of Medicine, 4Center for In Vivo Microscopy, Duke University Medical Center, 5KTM Research We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT. Behavior Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury Ashraf S. Gorgey1,2, Refka E. Khalil1, Robert M. Lester1, Gary A. Dudley3, David R. Gater4 1Spinal Cord Injury and Disorders Service, Hunter Holmes McGuire VAMC, 2Department of Physical Medicine and Rehabilitation, Virginia Commonwealth University, 3Deceased, Department of Kinesiology, The University of Georgia, 4Department of Physical Medicine and Rehabilitation, Penn State Milton S. Hershey Medical Center Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems. Neuroscience A Free-breathing fMRI Method to Study Human Olfactory Function Jianli Wang1, Sebastian Rupprecht1, Xiaoyu Sun1, Diana Freiberg1, Courtney Crowell1, Emma Cartisano1, Megha Vasavada1, Qing X. Yang1,2 1Center for NMR Research, Department of Radiology, Pennsylvania State University College of Medicine, 2Department of Neurosurgery, Pennsylvania State University College of Medicine We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing. Immunology and Infection Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity Mohammad Haque1, Kristin Fino1, Praneet Sandhu1, Jianxun Song1 1Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model. Biology Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production Sulei Xu*1, Xiang Li*1, Yuxin Liu2, Pingnian He1 1Department of Cellular and Molecular Physiology, College of Medicine, Penn State University, 2Lane Department of Computer Science and Electrical Engineering, West Virginia University Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels. Bioengineering Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents. Gregory M. Holmes1, Emily M. Swartz1, Margaret S. McLean1 1Department of Neural and Behavioral Sciences, Penn State University College of Medicine The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.