1 Characterizing Hearing in Mice ............................... 1
Karen P. Steel
1.1 Introduction ............................................. 1
1.2 Behavioral Tests of Hearing .............................. 2
1.3 Physiological Tests of Hearing ........................... 4
1.4 Anatomy of the Ear ....................................... 7
1.5 Conclusions ............................................. 12
Acknowledgements ........................................ 13
2 Molecular Phenotyping: Gene Expression Profiling ............ 15
Johannes Beckers
2.1 Why this Screen? Medical and Biological Relevance ....... 15
2.2 Examples: Diseases of Mouse and Man ..................... 17
2.3 Diagnostic Methods: History and State of the Art ........ 18
2.4 Technical Requirements for Screening Protocols (Short):
First and Second Line Approaches ........................ 20
2.5 Logistics (Whom, When, How Many, Why) ................... 20
2.5.1 Choice of Platform ................................ 20
2.5.2 Biological Samples ................................ 22
2.6 Trouble Shooting ........................................ 23
2.6.1 Preparation of Hybridization Target ............... 24
2.6.2 Critical Issues of Chip Hybridization ............. 27
2.6.3 Image Processing and Array Design ................. 30
2.7 Short-term Outlook ...................................... 31
3 Screening for Bone and Cartilage Phenotypes in Mice ......... 35
Helmut Fuchs, Thomas Lisse, Koichiro Abe, and
Martin Hrabé de Angelis
3.1 Introduction ............................................ 35
3.1.1 The Skeleton ...................................... 35
3.1.2 Skeletal Development in the Embryo ................ 36
3.1.3 Growth and Maintenance of Bone and Cartilage ...... 36
3.1.4 Diseases Involving Cartilage and Bone ............. 38
3.1.5 The Mouse as a Model for Skeletal Diseases ........ 41
3.2 Screening Protocols ..................................... 42
3.2.1 Morphological Analysis ............................ 42
3.2.1.1 Protocol .................................. 43
3.2.2 X-Ray Analysis .................................... 44
3.2.2.1 General ................................... 44
3.2.2.2 Imaging ................................... 45
3.2.2.3 X-Ray Analysis ............................ 45
3.2.2.4 Protocol .................................. 46
3.2.3 DXA- Analysis ..................................... 47
3.2.3.1 General ................................... 47
3.2.3.2 Advantages ................................ 48
3.2.3.3 Disadvantages ............................. 48
3.2.3.4 Small Animal Applications ................. 49
3.2.3.5 Precision and Accuracy .................... 49
3.2.3.6 Considerations ............................ 50
3.2.3.7 Protocol .................................. 51
3.2.4 Biochemical Bone Markers .......................... 51
3.2.4.1 Clinical Utility of Biochemical Markers
of Bone Turnover in Small Animals ......... 51
3.2.4.2 Mouse Markers of Bone Turnover/
Metabolism and Hormonal Regulation ........ 53
3.2.4.3 Variability/Sensitivity/Sample Choice ..... 58
3.2.4.4 Which Markers Should be Used During
the Screen? ............................... 58
3.2.5 Advanced Small Animal Imaging Techniques .......... 59
3.2.5.1 µQCT ...................................... 59
3.2.5.2 µCT ....................................... 61
3.2.5.3 µMRI ...................................... 63
3.2.5.4 Whole-mount Skeletal Preparations ......... 65
3.2.5.5 Histomorphometry .......................... 66
3.2.5.6 Miscellaneous ............................. 72
3.2.5.7 Order of the Tests ........................ 74
3.3 Conclusion .............................................. 76
List of Abbreviations ................................... 78
Appendix ................................................ 85
µCT Volumetric Data Processing .......................... 85
MRI Principles .......................................... 85
4 Clinical Chemical Screen .................................... 87
Martina Klempt, Birgit Rathkolb, Bemhard Aigner,
and Eckhard Wolf
4.1 Introduction ............................................ 87
4.1.1 Relevance of the Screen ........................... 87
4.1.2 Biology and Medical Application ................... 88
4.1.2.1 Biology of Clinical Chemical Parameters ... 88
4.1.2.2 Medical Application ....................... 88
4.2 Diseases in Mouse and Humans ............................ 89
4.2.1 Diagnostic Impact of Clinical Chemistry ........... 89
4.2.2 Clinical Chemistry in Selected Disorders .......... 91
4.2.2.1 Hypercholesterolemia ...................... 91
4.2.2.2 Albuminuria ............................... 91
4.2.2.3 Acute Myeloid Leukemia (AML) .............. 92
4.3 Clinical Chemistry as Diagnostic Tool ................... 93
4.3.1 History ........................................... 93
4.3.2 State of the Art .................................. 94
4.4 Technical Requirements and Screening Protocols .......... 94
4.4.1 Technical Requirements ............................ 94
4.4.4.1 Blood Collection .......................... 94
4.4.1.2 Sample Preparation ........................ 95
4.4.1.3 Sample Analysis ........................... 96
4.4.2 Screening Protocols ............................... 99
4.4.2.1 Primary Screen ............................ 99
4.4.2.2 Secondary Screen ......................... 100
4.4.2.3 Tertiary Screen .......................... 100
4.5 Logistics of the Screen ................................ 102
4.5.1 General Considerations ........................... 102
4.5.2 Lessons from ENU Mutants ......................... 103
4.6 Trouble Shooting ....................................... 104
4.6.1 Factors Interfering In Vivo ...................... 105
4.6.2 Factors Interfering In Vitro ..................... 105
4.7 Short-term Outlook ..................................... 105
5 Exploration of Metabolic and Endocrine Function in
the Mouse .................................................. 109
Marie-France Champy, Carmen A. Argmann, Pierre Chambon,
and Johan Auwerx
5.1 General Introduction ................................... 109
5.1.1 Investigating a Mouse with Endocrine and
Metabolic Dysfunction ............................ 109
5.1.2 Principles of Endocrine and Metabolic Testing .... 110
5.1.3 Strain in Relation to Mouse Models of Metabolic
Disease .......................................... 110
5.2 Evaluation of Energy Homeostasis ....................... 112
5.2.1 Body Weight and Food Intake ...................... 112
5.2.2 Energy Expenditure by Indirect Calorimetry ....... 114
5.2.3 Cold Test ........................................ 114
5.2.4 Exercise Test .................................... 115
5.2.5 Lean and Fat Composition of the Body ............. 116
5.3 Evaluation of Standard Clinical Chemistry Blood
Parameters ............................................. 117
5.4 Evaluation of Glucose Homeostasis ...................... 117
5.4.1 HOMA (Homeostasis Assessment Model) .............. 118
5.4.2 Meal Tolerance Test (MTT) ........................ 118
5.4.3 Intra-Peritoneal or Oral Glucose Tolerance Test
(IPGTT or OGTT) .................................. 118
5.4.4 Intra-Peritoneal Insulin Sensitivity Test
(IPIST) .......................................... 120
5.4.5 Glucose Clamps ................................... 120
5.4.6 Utilization of Glucose by Individual Tissues ..... 121
5.4.7 Insulin Secretion Test ........................... 122
5.5 Measurement of Serum Lipids and Lipoprotein
Parameters ............................................. 123
5.5.1 Serum Lipid Parameters ........................... 124
5.5.2 Isolation of Plasma Lipoprotein .................. 124
5.5.3 Apolipoproteins .................................. 225
5.6 Measurement of Hormones ................................ 126
5.7 Reproduction and Fertility ............................. 128
5.8 Bile Acids ............................................. 128
5.9 Post-Mortem Analysis and Histology ..................... 130
5.10 Molecular Imaging ..................................... 132
Acknowledgements ...................................... 132
6 Behavioral and Neurological Phenotyping in the Mouse ....... 135
Valter Tucci, Gonzalo Blanco and, Patrick M. Nolan
6.1 Introduction ........................................... 135
6.2 Human Neurological and Psychiatric Disorders ........... 136
6.2.1 Neurodegenerative Disorders ...................... 137
6.2.2 Mental Retardation Syndromes ..................... 138
6.2.3 Disorders Affecting Social Behavior .............. 139
6.2.3.1 Anorexia ................................. 139
6.2.3.2 Autism ................................... 139
6.2.4 Mood Disorders: Depression, Manias and
Schizophrenia .................................... 140
6.2.5 Anxiety .......................................... 141
6.2.6 Neuromuscular Disorders, Myopathies and
Neuropathies ..................................... 142
6.3 Behavioral and Neurological Phenotyping in the Mouse ... 143
6.3.1 Neurological and Neuromuscular Function .......... 144
6.3.2 Learning and Cognition ........................... 145
6.3.3 Social Behavior .................................. 146
6.3.4 Emotionality in Mice ............................. 147
6.3.5 Processing Sensory Information in Mice ........... 148
6.3.6 Endophenotypes ................................... 149
6.4 Behavioral and Neurological Screening Protocols in
the Mouse .............................................. 151
6.4.1 Screens for Neurological and Neuromuscular
Function ......................................... 153
6.4.1.1 Primary Screens .......................... 153
6.4.1.2 Secondary Screens ........................ 154
6.4.2 Screens for Motor Function ....................... 155
6.4.2.1 Primary Screens .......................... 155
6.4.2.2 Secondary Screens ........................ 156
6.4.3 Screens for Learning and Cognitive Function ...... 157
6.4.3.1 Primary Screens .......................... 157
6.4.3.2 Secondary Screens ........................ 157
6.4.4 Screens for Social Behavior ...................... 158
6.4.4.1 Primary Screens .......................... 258
6.4.4.2 Secondary Screens ........................ 159
6.4.5 Screens for Emotionality ......................... 160
6.4.5.1 Primary Screens .......................... 160
6.4.5.2 Secondary Screens ........................ 161
6.4.6 Screens for Central Processing of Sensory
Information ...................................... 162
6.4.6.1 Primary Screens .......................... 162
6.4.6.2 Secondary Screens ........................ 163
6.4.7 Supportive Screens ............................... 164
6.4.7.1 Biochemical Measurements ................. 164
6.4.7.2 Histopathology ........................... 164
6.4.7.3 Re-testing of Aged Mice .................. 165
6.5 Implementation of Behavioral and Neurological
Phenotypic Analysis .................................... 165
6.5.1 Gene-driven Approach (Reverse Genetics) .......... 165
6.5.2 Phenotype-driven Approach (Forward Genetics) ..... 166
6.5.3 Phenotype-driven Screens: A Short Guideline ...... 166
6.5.4 Environmental and Genetic Influences on Mutant
Behavior ......................................... 167
6.5.5 Standardization of Screening ..................... 168
6.6 Outlook ................................................ 168
6.6.1 Use of Imaging Technology ........................ 169
6.6.2 Investigation of Complex Traits in Compound
Mutants: Sensitized Screens ...................... 169
6.6.3 Use of Reporter Strains .......................... 170
7 Cardiovascular Disorders: Insights into In Vivo
Cardiovascular Phenotyping ................................. 177
Laurent Monassier and Andri Constant'mesco
7.1 Introduction ........................................... 177
7.2 In Vivo Imaging for Mouse Cardiovascular Phenotyping:
Interests and Limits ................................... 178
7.2.1 Echography ....................................... 178
7.2.2 Magnetic Resonance Imaging (MRI) ................. 178
7.2.3 Single Photon Emission Computed Tomography
(SPECT) .......................................... 182
7.2.4 Positron Emission Tomography (PET) Imaging ....... 183
7.2.5 X-Ray Computed Tomography (CT) ................... 184
7.2.6 Limitations in Studies Using Contrast Agents:
Particular Aspects of Nuclear-based Imaging ...... 185
7.3 Exploring the Heart in Living Animals .................. 187
7.3.1 Anatomy .......................................... 187
7.3.1.1 Systolic Function and Hemodynamics ....... 188
7.3.1.2 Global Systolic Function ................. 188
7.3.1.3 Regional Function ........................ 189
7.3.1.4 Diastolic Function ....................... 189
7.3.1.5 Impaired Myocardial Relaxation Pattern ... 190
7.3.1.6 Restrictive Filling Pattern .............. 190
7.3.1.7 Myocardial Perfusion, Metabolism and
Gene Expression Imaging .................. 190
7.3.1.8 Cardiac Conduction and Arrhythmias ....... 191
7.3.1.9 Exploring the Great Arteries ............. 193
7.3.1.10 Exploring Microvessels .................. 194
7.4 A Scheme for Identifying the Main Cardiovascular
Disorders in Genetically-modified Mice ................. 194
8 Phenotyping of Host-Pathogen Interactions in Mice .......... 201
Andreas Lengeling, Werner Müller, and Rudi Balling
8.1 Introduction ........................................... 202
8.2 Looking Back and Forward: History and
State-of-the-Art of Mouse Infection Phenotyping and
Studies of Genetic Infection Susceptibility ............ 202
8.3 The Impact of Mouse Genetics on the Understanding of
Human Infectious Diseases .............................. 205
8.4 Phenotyping at the GBF-Mouse Infection Challenge
Platform (ICP) ......................................... 207
8.4.1 Sreening Protocols ............................... 208
8.4.1.1 Infection with Listeria monocytogenes .... 208
8.5 Practical Guidelines ................................... 212
8.5.1 Growing Log-phase Cultures of Listeria
monocytogenes EGD for Mouse Infection ............ 212
8.5.2 Infection of Mice with Listeria monocytogenes
EGD .............................................. 212
8.5.3 Quantification of Bacterial Growth in Spleen
and Liver after L. monocytogenes Infection ....... 213
8.5.4 Troubleshooting .................................. 214
8.6 Outlook ................................................ 214
Acknowledgement ........................................ 215
9 Animal Models of Nociception ............................... 221
lldikó Rácz and Andreas Zimmer
9.1 Introduction ........................................... 221
9.2 Ethical Considerations ................................. 222
9.3 General Considerations ................................. 223
9.4 Assays for Acute Pain Thresholds ....................... 224
9.4.1 Thermal Stimuli .................................. 224
9.4.1.1 Tail-flick Test .......................... 224
9.4.1.2 Hargreaves Test .......................... 225
9.4.1.3 Hot-plate Test ........................... 226
9.4.2 Mechanical Stimuli ............................... 227
9.4.2.1 The Tail- and Paw-pressure Test .......... 227
9.4.2.2 Von Frey Filament Test ................... 227
9.5 Tonic and Visceral Pain Models ......................... 228
9.5.1 The Writhing Test ................................ 228
9.5.2 The Formalin Test ................................ 229
9.6 Hyperalgesia and Allodynia ............................. 230
9.6.1 Hyperalgesia and Allodynia Caused by
Neuropathic Pain ................................. 230
9.6.1.1 Chronic Constriction Injury Model ........ 231
9.6.1.2 Segmental Spinal Nerve Ligation Model .... 231
9.6.2 Hyperalgesia and Allodynia Caused by Tissue
Inflammation ..................................... 232
9.6.2.1 Determination of Mechanical Allodynia
and Thermal Hyperalgesia ................. 232
9.7 Stress-induced Analgesia ............................... 233
10 Mouse Phenotyping: Immunology .............................. 237
Svetosiav Kalaydjiev, Tobias J. Franz, and Dirk H. Busch
10.1 Introduction .......................................... 237
10.2 Diagnostic Methods .................................... 239
10.2.1 Antibody-based Techniques ...................... 239
10.2.2 Cellular Immunity Techniques ................... 240
10.2.3 Molecular Genetic Techniques ................... 241
10.3 Immunological Phenotyping at the German Mouse
Clinic ................................................ 242
10.4 Screening Protocols ................................... 245
10.4.1 FACS for Leukocyte Subpopulations .............. 246
10.4.1.1 Reagents and Equipment ................ 246
10.4.1.2 Procedure ............................. 246
10.4.2 Bead Array for Immunoglobulin Concentrations ... 247
10.4.2.1 Reagents and Equipment ................ 247
10.4.2.2 Procedure ............................. 247
10.4.3 ELISA for Autoantibodies ....................... 248
10.4.3.1 Reagents and Equipment ................ 248
10.4.3.2 Procedure ............................. 248
10.5 Troubleshooting ....................................... 249
10.6 Outlook ............................................... 249
11 Phenotyping Allergy in the Laboratory Mouse ................ 253
Thilo Jakob, Francesco Alessandrini, Jan Cutermuth,
Cabriele Köllisch, Anahita Javaheri, Antonio Aguilar,
Martin Mempel, Johannes Ring, Markus Ollert,
and Heidrun Behrendt
11.1 Introduction .......................................... 253
11.2 Phenotyping Different Forms of Allergic
(Hypersensitivity) Reactions .......................... 255
11.2.1 Immediate Type Hypersensitivity (Type I) ....... 256
11.2.1.1 Total IgE Baseline Levels in
Laboratory Mice ....................... 256
11.2.1.2 Allergen-specific IgE ................. 257
11.2.1.3 Passive Cutaneous Anaphylaxis (PCA) ... 259
11.2.1.4 Allergic Airway Inflammation, BAL,
Body Plethysmography .................. 262
11.2.1.5 Impact of Different Sensitization
and Challenge Protocols on
Parameters of Allergen-induced
Airway Inflammation ................... 265
11.2.2 Immune Complex Mediated Hypersensitivity
(Type III, Arthus Reaction) .................... 267
11.2.2.1 Reverse Passive Cutaneous Arthus
Reaction .............................. 268
11.2.3 Delayed Type Hypersensitivity (Type IV) ........ 270
11.2.3.1 Local Lymph Node Assay ................ 270
11.2.3.2 Contact Hypersensitivity Assay ........ 273
11.2.4 Granulomatous Hypersensitivity (Type V) ........ 275
11.2.4.1 Experimental Protocol for Type V
Hypersensitivity ...................... 275
11.3 General Considerations, Logistics and Outlook ......... 276
12 Eye Disorders .............................................. 283
Claudia Dalke, Oliver Puk, Angelika Neuhäuser-Klaus,
Jack Favor, and Jochen Craw
12.1 Introduction .......................................... 283
12.2 Medical and Biological Relevance of Eye Disorders ..... 283
12.3 Eye Disorders in Mouse and Man ........................ 284
12.3.1 Mutations Affecting Early Eye Development
Leading to Anophthalmia or Microphthalmia ...... 284
12.3.2 Cataracts ...................................... 285
12.3.3 Retinal Dysfunction and Degeneration ........... 286
12.3.4 Glaucoma ....................................... 286
12.4 Diagnostic Methods .................................... 287
12.4.1 History ........................................ 287
12.4.2 Routine Methods ................................ 288
12.4.2.1 Fundoscopy ............................ 289
12.4.2.2 Electroretinography (ERG) ............. 289
12.4.2.3 The Visual Tracking Drum or
Optomotor Drum ........................ 291
12.4.2.4 Measurement of Intraocular Pressure ... 292
12.4.2.5 Histological Analysis ................. 292
12.4.3 Methods in Development ......................... 292
12.4.3.1 The Scheimpflug Camera ................ 292
12.4.3.2 Length of the Axis .................... 293
12.4.3.3 Measurement of Intraocular Pressure ... 295
12.4.4 Future Combinations of First and Secondary
Screens for Vision Phenotyping ................. 296
12.5 Screening Protocols ................................... 296
12.5.1 Slit Lamp ...................................... 297
12.5.2 Funduscopy Using an Ophthalmoscope ............. 297
12.5.3 Electroretinography (ERG) ...................... 298
12.5.4 The Optokinetic Drum ........................... 300
12.6 Logistics ............................................. 301
12.6.1 Slit Lamp ...................................... 301
12.6.2 Ophthalmoscope ................................. 302
12.6.3 ERG ............................................ 302
12.6.4 The Optokinetic Drum ........................... 302
12.7 Troubleshooting ....................................... 302
12.7.1 Slit Lamp ...................................... 302
12.7.2 Ophthalmoscope ................................. 303
12.7.3 ERG ............................................ 303
12.7.4 The Optokinetic Drum ........................... 304
12.8 Outlook ............................................... 304
Acknowledgments ....................................... 304
13 EUMORPHIA and the European Mouse Phenotyping Resource for
Standardized Screens (EMPReSS) ............................. 309
Steve Brown, Heena Lad, Eain Creen, Ceorgios Ckoutos,
Hilary Gates, Martin Hrabé de Angelis,
and members of the EUMORPHIA consortium
13.1 Introduction .......................................... 309
13.2 The EUMORPHIA Project ................................. 309
13.2.1 Project Structure .............................. 310
13.3 Using Mouse Models .................................... 311
13.4 European Mouse Phenotyping Resource for Standardized
Screens (EMPReSS) ..................................... 312
13.4.1 Development of the SOPs ........................ 312
13.4.2 Review ......................................... 313
13.4.3 Validation of SOPs ............................. 314
13.5 Ontologies and Structure of the Empress Resource ...... 315
13.6 The EMPReSS Browser ................................... 316
13.7 Future Work ........................................... 317
Acknowledgments ....................................... 317
Index ......................................................... 321
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