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 References......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 Gerontological research is currently of great interest due to ever-increasing longevity of human life spans. Created to provide researchers and scientists with the technical methods essential to their work, Methods in Aging Research is a practical bench-top guide for everyone who plans, designs, or conducts research in aging. It highlights experimental procedures that are key to the investigation of the biological aging phenomena. Methods in Aging Research is a state-of-the-art reference that offers the most direct and clear cut descriptions available of methodological procedures used in aging research. Methods and protocols are critically evaluated and the advantages and disadvantages of each technique are explored. More than 70 expert contributors from around the world share their combined knowledge on a broad range of topics essential to everyone involved in cultivating and developing the gerontological research frontier