Studies of Aging PDF
Preview Studies of Aging
SPRINGER LAB MANUAL Springer Berlin Heidelberg New York Barcelona Hong Kong London Milano Paris Singapore Tokyo Hal Sternberg· Paola S. Timiras (Eds.) Studies of Aging With 37 Figures, one in Color Springer DR. HAL STERNBERG Vice President BioTime Inc. 935 Pardee St. Berkeley, CA 94710 USA PROF. DR. PAOLA S. TIMIRAS Department of Molecular and Cell Biology University of California Berkeley, CA 94720-3202 USA ISBN-13: 978-3-540-64600-6 e-ISBN-13: 978-3-642-59916-3 DOl: 10.1007/ 978-3-642-59916-3 Library of Congress Cataloging-in-Publication Data Studies of aging 1 Hal Sternberg, Paola S. Timiras (eds.). p. cm. - (Springer lab manual) Includes bibliographical references and index. 1. Aging-Research-Methodology. 2. Aged-Diseases-Research-Methodology. 3. Geriatrics- Methodology. l. Sternberg, Hal, 1953- . II. Timiras, Paola S., 1923- . III. Series. QP86.S78 1999 612.6'7'0724 - dc21 99-20522 CIP This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfIlms or in any other way, and storage in data banks. Du plication of this publication or parts thereofis permitted only under the provisions ofthe German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copy right Law. © Springer-Verlag Berlin Heidelberg 1999 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such in formation by consulting the relevant literatljre. Cover design: design & production GmbH, Heidelberg Typesetting: Mitterweger Werksatz GmbH, Plankstadt SPIN: 10547915 27/3137 - 543 2 1 0 - Printed on acid free paper Preface Although experimental gerontology has been a recognized field of study for over 40 years, there is a dearth of texts which have concentrated on experimental techniques and procedures of particular use to gerontologists. This is partly because most of these techniques are not uniquely used by gerontologists. Nevertheless, certain techniques are used repeatedly in geronto logical studies, and many of these are effectively brought to gether in this volume. Early research on aging was characterized by attempts to ob tain support for various theories of aging, but most of the studies were descriptive and correlative in nature. It has become increas ingly clear that the various theories can be coalesced around two kinds of factors: genetic and environmental. Among the envir onmental factors the one most often studied has been oxidative damage, because oxidative damage is ubiquitous and continuous in almost all cells, coming primarily from leakage of superoxide anion from the mitochondria during electron transport. Thus, measurements of oxidative damage to cellular and extracellular macromolecules (especially DNA, proteins and lipids), and de termination of levels of protective antioxidants, have been and remain an important focus in gerontological research (Chapters 6 and 7). Oxygen radicals are a major cause of DNA damage, leading to the formation of a wide variety of modified bases. De letions may occur either during the repair of damaged DNA or during DNA replication, and these events are particularly easy to detect, although difficult to quantitate accurately, in mitochon drial DNA by PCR analysis (Chapter 1). The level of deletions increases with age, especially in muscle and brain tissue, and may even be a biomarker of aging. Furthermore, PCR analysis provides the ability to detect even very rare mutations through genotypic selection, in addition to phenotypic selection. 'II Preface Unfortunately, reliable biomarkers of human aging are yet to be developed. The results from the Baltimore Longitudinal Study of Aging have demonstrated the remarkable plasticity of human aging, which is presumably due to both genetic and environmen tal factors. Thus, the most reliable measure of aging, although not the easiest to implement, remains the survival curve, and survival curves have been used to characterize different aging rates in rodents, as well as nematodes and insects (Chapter 9). One particular advantage of insects such as fruit flies, is the opportunity to use extremely large cohorts. Thus, it is pos sible in such studies to examine the properties and behavior of the very last few survivors (the very oldest old) from a population of a million or more individuals. Survival curves have also been extensively used to verify the extension of maximum life span by restriction of caloric intake (Chapter 11), an intervention which has retarded aging in every animal species where it has been ade quately tested. It is particularly important that the diets used re strict only calories, while providing all the essential nutrients and vitamins, and well-tested diets for this purpose are provided here. Survival curves will often need to be done to confirm the validity of any putative life-extending intervention, as these interventions will have to be well tested in animal model systems before application to humans. Thestudyofagingalsoincludesthestudyofage-relateddisease. Alzheimer's disease (AD) can be studied non-invasivelyin human brain by imaging using X-ray CT, SPECT, PET and magnetic re sonance (Chapter 14), and in animal models (Chapters4 and 10) by a variety of invasive techniques. These latter techniques include the use ofm ouse hippocampal slices and the infusion ofA pep tides into rat brain. Similarly, cardiovascular disease can be studied by investigating the characteristics of isolated vascular smooth mus cle cells (Chapter 5), because of the strong correlation between migration oft hese cells and vascular injury. In laboratoryrodents, and probably in primates and humans as well, stress often repre sents an unwanted side-effect ofe xperimental manipulations, e.g. as in dietary restriction. Therefore, some of the manifestations of stress, such as activation of the hypothalmus-pituitary-adreno cortical axis should be evaluated, and more importantly, efforts should be made to use techniques that minimize the impact of stress on aging processes. Some of these techniques for assessing and minimizing stress in rats are discussed in Chapter 12. Preface VII One aspect of aging is the decline in tissue and systemic home ostasis. This includes the proper number and balance of different kinds of cells. Thus, it is important to understand the role of both cell replication and cell death in maintaining this balance, and how aging affects these processes. Chapter 2 not only describes methods for following population doublings and assessing the senescent state of cells in vitro, but also provides an innovative assay for detecting senescent cells in vivo. In contrast, Chapter 3 describes techniques for determining whether cells are being lost by apoptosis, as it is becoming clear that apoptosis plays a more important role in many biological processes, including aging, than previously thought. If too many cells are lost during aging in a particular tissue or organ, transplantation of cells, tissues or organs might provide a logical intervention. Some of the tech niques related to organ transplantation are presented here in Chapter 13. Finally, a critical factor in any study using an animal model is the choice of an appropriate animal model. Chapter 8 addresses the many factors that must be considered when choosing an an imal species and strain to use as a surrogate for human aging. Making the right choice may be the most important factor in the success of the research, as the value of any model depends on the context of the specific research agenda proposed. Thus, this book presents many useful concepts and experi mental details for the study of aging and age-related disease in humans and a variety of animal model systems. Huber R. Warner, Ph.D., Biology of Aging Program, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 Contents Part A Molecular Methods Introduction to Molecular Methods HAL STERNBERG 1 Chapter 1 Genotypic Selection of Age-Related DNA Rearrangements by PCR ALICE WONG AND GINO CORTOPASSI .........•.•...•• 3 Chapter 2 Measurement of Antioxidants in Cutaneous Tissues J.J. THIELE, J.K. LODGE, J.H. CHOI AND 1. PACKER ...... 15 Subprotocol 1: Skin Separation and Extraction. . . . . . . . . . 16 Subprotocol 2: Lipophilic Antioxidants: Alpha-Tocopherol and Ubiquinol ....... 18 Subprotocol 3: Hydrophilic Antioxidants I: Ascorbate and Urate. . . . . . . . . . . . . . . . . . . 22 Subprotocol 4: Hydrophilic Antioxidants II: Glutathione 25 Subprotocol 5: Antioxidant Enzymes - Catalase Activity 28 Subprotocol 6: Antioxidant Enzymes - Glutathione Peroxidase Activity . . . . . . . . . 30 X Contents Part B Cellular Methods Introduction to Cellular Methods HAL STERNBERG ....................•....••......... 33 Chapter 3 Cellular Aging / Replicative Senescence JUDITH CAMPISI .................................... 35 Chapter 4 Methods for Evaluating a Slice Culture Model of Alzheimer's Disease M.E. HARRIS-WHITE, J.J. SIGEL, S.A. FRAUTSCHY, GREG M. COLE ...................................... 46 Chapter 5 Cell Death RICHARD A. LOCKSHIN .............................. 58 Chapter 6 Method for Studying the Migration of Aging Vascular Smooth Muscle Cells ROBERT E. MONTICONE, CLAUDIO BILATO, MICHAEL T. CROW ............•..................... 78 Chapter 7 Sensitized Chemiluminescence and Fluorescence Methods in Studies of Oxidative Stress VICTOR S. SHAROV, KARLIS BRIVIBA AND HELMUT SIES 90 Part C Animal Models Introduction to Animal Models HAL STERNBERG 103 Chapter 8 How to Choose An Animal Model RICHARD L. SPROTT ................................. 105 Contents XI Chapter 9 Measuring Mortality and Reproduction in Large Cohorts of the Mediterranean Fruit Fly JAMES R. CAREY AND PABLO LIEDO ................... 111 Subprotocol 1: Individuals and/or Pairs .............. " 113 Subprotocol 2: Grouped Cages ........................ 116 Part D Physiological Methods Introduction to Physiological Methods HAL STERNBERG .................................... 125 Chapter 10 Methods for Evaluating in Vivo Rodent Models for Alzheimer's Disease S.A. FRAUTSCHY, J.J. SIGEL, M.E. HARRIS-WHITE, T. CHU AND G.M. COLE 127 Chapter 11 Dietary Restriction in Aging B.J. MERRY ......................................... 143 Chapter 12 Methods for the Evaluation of the Hypothalamo-Pituitary Adrenoccortical Axis in the Aged Rat SERGIO SCACCIANOCE, RAFFAELLA NICOLAI, DANIELA AFFRICANO, KA TIA LOMBARDO AND LUCIANO ANGELUCCI ........................... 164 Chapter 13 Assessing Blood Replacement Fluids for Organ Preservation and Low Temperature Surgery HAL STERNBERG, PAUL SEGALL, HAROLD WAlTZ AND JUDITH SEGALL ................................. 171 Chapter 14 Brain Imaging in Normal and in Alzheimer's Disease T.F. BUDINGER ...................................... 182 Subject Index ....................................... 207
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