Loma Linda University View Institution's Website 11 articles published in JoVE Biology Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma Md A. Hakim1, Paulo W. Pires2, Erik J. Behringer1 1Department of Basic Sciences, Loma Linda University, 2Departments of Physiology, Surgery and Neurosurgery and Sarver Heart Center, University of Arizona College of Medicine Tucson Intensive preparation of intact mouse cerebral endothelial "tubes" from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential. Biology Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method Gina D. Roque-Torres1, Nina C. Nishiyama2, Seta Stanbouly2, Xiao Wen Mao2 1Center for Dental Research, Loma Linda University, 2Department of Basic Sciences, Division of Biomedical Engineering Sciences (BMES), Loma Linda University We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage. Immunology and Infection In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction Hongzheng Bi1,2, Samiksha Wasnik1, David J. Baylink1, Chenfan Liu1,3,4, Xiaolei Tang1,3 1Division of Regenerative Medicine, Department of Medicine, Loma Linda University, 2Zhengzhou University, 3Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, Long Island University, 4Jinan Infectious Disease Hospital, Shandong University Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo. Neuroscience Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice Samantha B. Palahnuk1,2, Jonathan A. Abdala1, Vadim V. Gospodarev3, Christopher G. Wilson1 1Center for Perinatal Biology, Department of Basic Sciences, Loma Linda University, 2Department of Biology, The College of New Jersey, 3Department of Neurosurgery, Loma Linda University This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem. Biology Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium Md A. Hakim1, Erik J. Behringer1 1Department of Basic Sciences, Loma Linda University Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables. Immunology and Infection Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein Yi Xu*1, Samiksha Wasnik*1, David J. Baylink1, Edmundo Carreon Berumen1, Xiaolei Tang1 1Department of Medicine, Division of Regenerative Medicine, Loma Linda University Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein. Medicine Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line Jacqueline S. Coats1, Ineavely Baez1, Cornelia Stoian1, Terry-Ann M. Milford2, Xiaobing Zhang3, Olivia L. Francis1, Ruijun Su1, Kimberly J. Payne1 1Department of Pathology and Human Anatomy, Loma Linda University, 2Department of Basic Sciences, Loma Linda University, 3Department of Medicine, Loma Linda University Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models. Developmental Biology Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors Wei Wen1, Jian-Ping Zhang1, Wanqiu Chen2, Cameron Arakaki2, Xiaolan Li1,2, David Baylink2, Gary D. Botimer3, Jing Xu1, Weiping Yuan1, Tao Cheng1,4,5,6,7, Xiao-Bing Zhang1,2 1State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Division of Regenerative Medicine, Department of Medicine, Loma Linda University, 3Department of Orthopaedic Surgery, Loma Linda University, 4Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, 5Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, 6Collaborative Innovation Center for Cancer Medicine, 7Tianjin Key Laboratory of Blood Cell Therapy and Technology This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Medicine Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase Paul R. Krafft1, William B. Rolland1, Kamil Duris1, Tim Lekic1, Aaron Campbell2, Jiping Tang1, John H. Zhang1,3,4 1Department of Physiology and Pharmacology, Loma Linda University School of Medicine, 2College of Natural and Agricultural Sciences, University of California, Riverside, 3Department of Anesthesiology, Loma Linda University School of Medicine, 4Department of Neurosurgery, Loma Linda University School of Medicine Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice. Medicine The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness Hani H. Al-Nakhli1, Jerrold S. Petrofsky1,2, Michael S. Laymon2, Lee S. Berk1 1Loma Linda University, 2Azusa Pacific University The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising. Medicine Biochemical Measurement of Neonatal Hypoxia Megan S. Plank1, Teleka C. Calderon1, Yayesh Asmerom2, Danilo S. Boskovic1, Danilyn M. Angeles2 1Division of Biochemistry, Department of Basic Sciences, Loma Linda University, 2Division of Physiology, Department of Basic Sciences, Loma Linda University A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).