Methods in aging research

Contents......Page 4
Preface......Page 7
Section A: General Theoretical Approaches to Aging Research......Page 14
1. Population Study of Mortality and Longevity with Gompertzian Analysis......Page 15
1.2.2 Construction and Interpretation......Page 16
1.2.3 Additional Life Table Parameters and Relationships......Page 17
1.3.2 The Force of Mortality......Page 22
1.3.3 Smoothing Age-Specific Mortality Rates......Page 23
1.4.1 Common Parametric Models......Page 24
1.4.2 Fitting Mortality Data to Models......Page 27
1.5.3 Density Effects......Page 32
1.5.6 Visualizing Cohort Survival and Individual Reproduction......Page 33
References......Page 34
2.1 Introduction......Page 37
2.3.1 Major Features and Advantages......Page 38
2.3.2 Precautions......Page 40
2.4.2 Examples of Longitudinal Studies......Page 42
2.4.3 Disadvantages and Limitations......Page 44
2.5.1 Major Features and Advantages......Page 46
2.5.2 Modified Sequential Designs and Time-Lag Studies......Page 47
2.6.2 Health Issues......Page 49
2.7 Nonhuman Animal Studies......Page 50
2.8 Summary......Page 51
References......Page 52
3. Mathematical and Computational Tools for Gerontological Research......Page 55
3.1.1 Philosophy and Phenomenology......Page 56
3.2.1 Visualization Philosophy......Page 57
3.2.4 Data Analysis......Page 60
3.3 Chemistry and Pharmacology /Pharmacokinetics......Page 62
3.4.2 Sequencing......Page 63
3.5 Cell Biology, Physiology, and Metabolism......Page 64
3.6.2 Cardiovascular Dynamics......Page 65
3.6.6 Patient-Based Physiological Simulation......Page 66
3.8 Dentistry and Oral Physiology......Page 67
3.9.2 Space......Page 68
3.10 Closing Thoughts......Page 69
References......Page 70
Section B: Experimental In Vitro Models in Aging Research......Page 87
4. Use of the Fibroblast Model......Page 88
4.1 Introduction......Page 89
4.3 Importance to Current Research......Page 90
4.3.2 Strengths: In Vivo/In Vitro Parallels......Page 91
4.3.3 Limitations / Considerations / Caveats of the System......Page 93
4.4 Cellular and Molecular Markers of In Vitro Senescence......Page 94
4.4.2 Changes in Macromolecular Synthesis and Content......Page 95
4.4.3 Cell Cycle Position of Arrested Cells......Page 99
4.4.4 Signal Transduction Pathways......Page 100
4.5 Propagation of Human Fibroblasts: Materials and Methods......Page 102
4.5.2 Preparation of Solutions and Other Items Required for Procedures......Page 103
4.5.3 Cell Culture Procedures......Page 110
4.5.4 Replicative Life Span......Page 112
4.5.6 Thymidine Incorporation......Page 113
4.6 Summary and Conclusions......Page 115
References......Page 117
5. Electrophysiological Assessment of the Aged Hippocampus In Vivo and In Vitro......Page 126
5.1.2 Role of Electrophysiological Studies in Aging Research......Page 127
5.2.1 Brief History of Hippocampal Electrophysiology......Page 128
5.2.3 Choice of Experimental Subject......Page 129
5.3.1 In Vivo Recording......Page 130
5.3.2 In Vitro Recording......Page 136
5.4.1 Hippocampal Electrophysiology In Vivo......Page 140
5.4.2 Hippocampal Electrophysiology In Vitro......Page 145
References......Page 149
6.1 Introduction......Page 155
6.2.1 Earlier Genetical Analysis in Biogcrontology......Page 157
6.2.3 Recent Examples of the Genetical Approach to Biogerontology......Page 158
6.3 General Descriptions of Genetic Tools and Techniques Needed......Page 161
6.4.1 Selection Experiments......Page 163
6.4.2 Quantitative Trait Loci QTL Mapping......Page 164
6.4.3 Multiple Gene Mutagenesis......Page 165
6.4.4 Single Gene Mutagenesis......Page 166
6.4.6 Manipulation of Environmental Variables......Page 169
References......Page 170
7. Experimentation with Nematodes......Page 176
7.1.1 General Background......Page 177
7.1.2 General Description......Page 178
7.1.3 The Life Cycle and Morphological Features......Page 179
7.1.4 Developmental Biology......Page 180
7.2.1 The Use of Turbatrix Aceti in the 1970s: Advantages of the System......Page 182
7.2.2 Emergence of the Concept of Accumulation of Altered Enzyme Molecules with Age......Page 183
7.2.3 Discovery of the Decline in Function of the Protein Degradation System......Page 184
7.3.1 Choice of Species and Strains, and of Either Axenic or Monoxenic Culture Systems......Page 185
7.3.2 Synchronization and Maintenance of Cultures of Organisms of Uniform Age......Page 188
7.3.3 Biochemical Studies: Enzymes, Nucleic Acids, Protein Labeling, and Degradation Studies......Page 189
7.3.4 Developmental Studies and Dauer Larva Development......Page 191
7.4.1 Impact and Relevance of Nematode Studies on the Field of Aging Research......Page 193
7.4.2 Evaluation of the Main Merits and Disadvantages of the Nematode Model......Page 194
References......Page 196
8. Experimentation with the Yeast Model......Page 200
8.1 Introduction......Page 201
8.2.2 Microneedles......Page 202
8.2.4 Maintenance of Strains......Page 204
8.2.6 Procedure......Page 205
8.3.1 Separation of Age-Synchronized Cells by Rate-Zonal Sedimentation in Sucrose Gradients......Page 207
8.3.2 Sampling of Cells from Mixed Populations......Page 214
8.3.3 Other Methods......Page 218
References......Page 221
Section C: Vertebrate Models in Aging Research......Page 223
9.1 Introduction......Page 224
9.1.1 Interagency NIA/NCTR Study......Page 225
9.2.1 History......Page 228
9.2.2 Current Methods for Rearing Aging Rodents......Page 231
9.3.1 Facilities, Design, and Maintenance......Page 232
9.3.4 Pathology and Histopathology Support......Page 233
9.3.5 Animal Care Support......Page 235
9.3.6 Diet Preparation Support......Page 236
9.3.7 Computer Support......Page 237
9.4.1 Isolators......Page 238
9.5 Conclusions......Page 239
References......Page 240
10. Choice of Rodent Model for Aging Research......Page 243
10.1.2 Rodents Used......Page 244
10.2.3 Negative Impact of Extensive Use of Rodents......Page 246
10.3.1 Requirements Common to All Gerontologic Studies......Page 247
10.4.1 Detailed Definition of the Study Design......Page 250
10.4.3 Meeting the Gerontologic Requirements......Page 251
References......Page 252
11. Aging Experiments Using Nonhuman Primates......Page 255
11.2.2 Longevity......Page 256
11.3.2 Primates......Page 257
11.4.1 Availability......Page 258
11.4.3 Diet and Nutritional Needs......Page 260
11.5.1 Nervous System and Behavior......Page 261
11.5.2 Endocrine System......Page 263
11.5.4 Immune System......Page 264
11.6.1 Dietary Restriction......Page 265
11.6.2 Biomarkers......Page 266
References......Page 267
Section D: Methods of Assessing Aging Processes......Page 274
12. Dietary Restriction......Page 275
12.1 Introduction......Page 276
12.2.1 DR by Controlled Growth or Body Weight......Page 278
12.2.2 DR by Predetermined Restriction of Food Allotment......Page 289
12.2.4 Degree and Initiation of Restriction......Page 291
12.3.1 Macronutrients......Page 292
12.3.3 Dietary Sources......Page 293
12.4.1 Species, Strain, and Gender......Page 294
12.5.1 Animals......Page 295
12.5.3 Diet......Page 297
12.5.4 Feeding Regimen......Page 298
12.5.5 Rat Population and Usage......Page 299
12.6 Summary......Page 300
References......Page 301
13.1.1 Research Issues in Geriatric Nutrition......Page 305
13.2.2 In a Community Setting......Page 308
13.3 Determination of Nutritional Status......Page 310
13.3.1 Dietary Assessment......Page 312
13.3.2 Anthropometric Assessment......Page 313
13.3.3 Laboratory Measurements......Page 316
13.4.1 Allocation of Treatment Regimen......Page 317
13.4.2 Intervention......Page 318
13.6 Statistical Analyses......Page 319
Re feren ces......Page 320
14.1 Introduction......Page 325
14.2.1 Morbidity, Mortality, and Exercise......Page 326
14.2.3 Degenerative Disease and Aging......Page 327
14.2.4 Exercise and Free Radical Theory of Aging......Page 328
14.3.1 General Considerations......Page 329
14.3.2 Levels of Investigation......Page 330
14.3.3 Major Factors Determining Exercise Response......Page 331
14.3.4 interactions of Exercise and Other Biological Factors......Page 335
References......Page 339
15.1 Introduction......Page 344
15.2.1 Regulation by Presynaptic Autoreceptors and the Effect of Age......Page 346
15.2.3 Regulation of Intracellular Signaling Systems, Exocytosis and the Effect of Age......Page 347
15.3 General Design......Page 349
15.4.1 Preparation of Brain Slices and Use in a Superfusion System......Page 351
15.4.2 Preparation and Superfusion Procedure for Brain Synaptosomes......Page 352
15.4.3 Use of Cardiac Synaptosomes to Examine Age-Related Changes in Norepinephrine Release in the Heart......Page 353
15.4.4 Calculation......Page 355
15.5 Conclusion......Page 357
References......Page 358
16. Pathological Analysis in Aging Research......Page 363
16.1 Introduction......Page 364
16.2.1 Historical Perspectives......Page 365
16.2.2 Importance of Aging Research......Page 366
16.3 General Description and Principles and Techniques......Page 367
16.3.1 Materials and Methods......Page 368
16.3.2 Laboratory Requirements......Page 370
16.3.4 Experin1ental Design......Page 371
16.4.1 Necropsy......Page 372
16.4.2 Histology......Page 377
16.4.3 Special Techniques......Page 378
16.4.4 Microscopic Evaluation......Page 379
\rReferences......Page 383
Section E: Molecular and Evolutionary Probes of Senescence Alterations......Page 387
17. Transgenic Manipulation of the Mouse Genome......Page 388
17.2 Historical Perspectives......Page 389
17.3.1 Embryo Collection, Pronuclear Microinjection, and Embryo Transfer......Page 390
17.3.2 Mice Needed for Transgenic Founder Mouse Production......Page 393
17.3.3 Transgene Design and Preparation for Microinjection......Page 394
17.3.4 Identification of Transgenic Mice......Page 397
17.4.1 Preparation of Transgene DNA for Microinjection......Page 399
17.4.2 Vasectomized Stud Male Mice and Pseudopregnant Female Mice......Page 400
17.4.3 Fertile Stud Male Mice and Superovulated Female Donor Mice......Page 401
17.4.4 Embryo Retrieval and Preparation for Microinjection......Page 402
17.4.5 Pronuclear Microinjection of One-Cell Embryos......Page 405
17.4.6 Transfer of Injected Embryos to Pseudopregnant Females......Page 407
17.4.7 Isolation of DNA from Mouse Tail Biopsies......Page 409
17.5.1 Establishing Transgenic Mouse Lines......Page 410
17.5.3 Establishing Lines Homozygous for the Transgene......Page 412
17.5.5 Cryopreservation of Transgenic Lines......Page 413
References......Page 415
18.1 Introduction......Page 417
18.2 Background and Historical Perspective......Page 418
18.3.1 DNA Blotting......Page 419
18.3.2 TRF Length Analysis......Page 421
18.3.3 Fluorescence in situ Hybridization FISH......Page 426
18.4 Applications of Telomere Length Analysis......Page 431
18.5 Summary and Conclusions......Page 432
References......Page 433
19.1 Introduction......Page 436
19.2.1 Historical Use of Comparative Method in Aging Research......Page 437
19.2.2 Potential Problems in the Comparative Method......Page 438
19.3.1 Formulating and Evaluating Hypotheses about Aging Mechanisms......Page 441
19.3.2 Assessing Generality of Specific Aging Mechanisms......Page 442
19.3.3 Identifying Key Aging Mechanisms from an Array of Candidates......Page 443
19.4 Example: Comparative Assessment of Metabolism and Body Temperature as Contributors to the Anti-aging Effect of Caloric Restriction......Page 445
19.5 Conclusions......Page 448
References......Page 449
20.1 Introduction......Page 452
20.1.1 Necrosis and Apoptosis......Page 453
20.1.2 Definitions of Apoptosis and Programmed Cell Death......Page 454
20.1.3 Senescence and Differentiation to Death......Page 455
20.1.6 The Genetic Basis of Apoptosis and Programmed Cell Death......Page 456
20.2.1 Histological Staining of Apoptosis......Page 457
20.2.2 Fine Structural Changes......Page 459
20.3.1 Electrophoresis......Page 461
20.3.2 Cytochemistry......Page 463
20.3.3 Cell Viability Assay......Page 466
20.4 Flow Cytometric Assays......Page 467
20.5 Non-Apoptotic Programmed Cell Death......Page 468
References......Page 469
21. Mitochondrial DNA Deletions......Page 473
21.1 Introduction......Page 474
21.2 Background......Page 475
21.3.2 Buffers and Media......Page 476
21.3.3 Isolation of Total DNA From Different Tissues......Page 477
21.3.4 Isolation of DNA From Mitochondria of Different Tissues......Page 479
21.4.1 In Situ Hybridization......Page 480
21.4.2 Identification of Mitochondrial DNA Deletions by Standard PCR......Page 481
21.4.3 Long Extension PCR......Page 489
21.4.4 Single Fiber PCR......Page 490
21.4.5 Quantitative PCR......Page 493
21.5 Conclusions......Page 503
References......Page 504
Section F: Techniques for Exploring Age-Related Intra- and Subcellular Changes......Page 510
22. Intracellular Signal Transduction Pathways Involved in Hepatocyte DNA Synthesis Following Growth Factor Stimulation......Page 511
22.1.2 Proliferative Response in Young and Aged Cells......Page 512
22.2.1 Hepatocyte Isolation and Culture......Page 515
22.2.2 DNA Synthesis Measurements......Page 517
22.2.3 Cyelin- Dependent Kinase 2  CDK2 Assay......Page 518
22.2.4 ERK Mitogen- Activated Protein Kinase Assay......Page 520
22.2.5 p70 56 Kinase Assay......Page 522
22.3.2 Age- Associated Impairment in the Early Signaling Pathways......Page 524
22.3.3 Future Directions......Page 525
References......Page 526
23.1 Introduction......Page 528
23.2 Background......Page 529
23.3 Isolation of Mitochondria......Page 531
23.4.1 Procedure......Page 532
23.4.2 Validity......Page 533
23.4.3 State 4 and State 3 H202 Production......Page 534
23.4.4 H2O2 Pulse Experiments......Page 535
23.4.5 Standards......Page 537
23.4.6 Precautions......Page 538
23.5 Localization of the Oxygen Radical Source......Page 539
References......Page 541
24. Methods for the Study of Immune Cells in Aging......Page 544
24.1 Introduction......Page 545
24.2.2 Measurement of Lymphocyte Response to Mitogens......Page 546
24.2.4 Measurement of T Cell Subsets Effector Function......Page 547
24.2.5 Measurement of B Cell Function......Page 548
24.3.2 ELISPOT Assay......Page 549
24.3.3 Sandwich ELISA Assay for Detection and Quantitation of Cytokines......Page 550
24.4.1 Semi-Quantitative Polymerase Chain Reaction PCR Technique......Page 551
24.4.2 Semi-Quantitative RT-PCR Methods......Page 552
24.4.3 Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis......Page 553
24.5.1 Enumeration of Lymphocyte Population by Antibodies Specific for Cell-Surface Molecules......Page 556
24.5.3 Measurement of Apoptosis......Page 558
References......Page 559
Section G: Techniques for Assessing Age-Related Oxidative Modification......Page 563
25. Lipid Peroxidation......Page 564
25.2.1 Formation of Lipid Peroxides......Page 566
25.2.2 Isomerization of Lipid Peroxides......Page 570
25.2.3 Decomposition of Lipid Peroxides......Page 571
25.3.2 Hydrogen Abstraction for the Initiation of Lipid Peroxidation......Page 574
25.3.3 Antioxidants......Page 575
25.4.1 Preparative Procedures......Page 576
25.5 Analysis of Lipid Peroxides......Page 578
25.5.1 Spectral Analysis......Page 579
25.5.2 Chromatographic Analysis......Page 580
25.5.3 Chemical and Enzymatic Analysis......Page 582
25.6.1 Indices of Lipid Peroxide Formation......Page 583
25.6.2 Indices of Lipid Peroxide Decomposition......Page 588
Concluding Remarks......Page 596
References......Page 597
26.1 Introduction......Page 600
26.3.1 Materials and Methods......Page 601
26.3.2 Analysis of DNA Modifications......Page 602
26.4.1 Identification Using Selected-Ion Monitoring......Page 605
26.4.2 Quantification......Page 606
26.6 Limitations and Comments......Page 609
References......Page 611
27.1 Introduction......Page 614
27.2 Background: Use of Transgenic Mice for Mutation Analysis in vivo......Page 616
27.3 System Description......Page 617
27.4.1 Transgenic Animals......Page 619
27.4.3 Reagents......Page 620
27.4.5 Tissue Collection and DNA Extraction......Page 621
27.4.7 Magnetic Bead Rescue of LACZ Plasmid from Mouse Genomic DNA......Page 622
27.4.9 Mutant Characterization......Page 623
27.5.2 Validity of the Plasmid Model in Detecting Genome Rearrangements......Page 624
27.5.3 Relevance of the Model for Age-Related Mutation Accumulation......Page 625
References......Page 626
28.1 Introduction......Page 629
28.2 Background......Page 630
28.3.1 Tritiated Borohydride Method......Page 631
28.3.2 2,4-0NPH Extraction Method......Page 632
28.3.3 2,4-0NPH Oerivatization Filtration Method......Page 633
28.3.4 2,4-0NPH HPLC Method......Page 634
28.3.5 S0S Oerivatization Methods for HPLC and Immunoblotting......Page 636
28.3.6 Fluorescein Thiosemicarbazide for Gel Electrophoresis......Page 638
28.4.1 HPLC Following Proteolytic Digestion......Page 639
28.4.2 Stable Isotope GC/MS......Page 640
28.5 Protein Oxidation Secondary To Lipid Peroxidation......Page 642
28.6 Conclusion......Page 644
References......Page 645
29.1 Introduction......Page 648
29.2.1 General Methods......Page 655
29.2.3 Furosine Assay by HPLC......Page 656
29.2.5 Borohydride Reduction and Labeling of Amadori Products with 3H-NaBH4......Page 658
29.3 Glycoxidation Products......Page 659
29.3.1 Carboxymethyllysine Assay by HPLC with Post- Column Detection......Page 660
29.3.3 Pentosidine Assay by HPLC......Page 661
29.4.2 Immunoreactive \"AGE\" ELISA......Page 663
29.4.3 Carboxymethyllysine by ELISA......Page 664
29.4.4 Pentosidine by ELISA......Page 665
References......Page 666
Index......Page 673
A......Page 674
D......Page 675
E......Page 677
I......Page 679
L......Page 680
M......Page 682
N......Page 683
P......Page 684
S......Page 685
Y......Page 686