Table Of ContentSPRINGER
LAB MANUAL
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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
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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
