Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 Monitoring nociception by analyzing gene expression changes in the central nervous system of mice Asner, I N Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-46678 Dissertation Originally published at: Asner, I N. Monitoring nociception by analyzing gene expression changes in the central nervous system of mice. 2010, University of Zurich, Vetsuisse Faculty. Monitoring Nociception by Analyzing Gene Expression Changes in the Central Nervous System of Mice Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Igor Asner von St. Cergue VD Promotionskomitee Prof. Dr. Peter Sonderegger Prof. Dr. Kurt Bürki Prof. Dr. Hanns Ulrich Zeilhofer Dr. Paolo Cinelli (Leitung der Dissertation) Zürich, 2010 Table of contents Table of content Curriculum vitae 6 Publications 9 Summary 11 Zusammenfassung 14 1. Introduction 17 1.1. Pain and nociception 17 1.1.1 Nociceptive neurons and Mechanoceptors 18 1.1.2 Activation of the nociceptive neurons at the periphery 21 1.1.2.1 Response to noxious heat 22 1.1.2.2 Response to noxious cold 23 1.1.2.3 Response to mechanical stress 24 1.1.3 Nociceptive message processing in the Spinal Cord 25 1.1.3.1 The lamina I and the ascending pathways 25 1.1.3.2 The lamina II and the descending pathways 26 1.1.4 Pain processing and integration in the brain 27 1.1.4.1 The Pain Matrix 27 1.1.4.2 Activation of the descending pathways 29 1.1.5 Inflammatory Pain 31 1.2. Genes involved in pain mechanisms 34 1.2.1. Transgenic mice and pain 34 1.2.2. Pain genes databases 36 1.2.3. Assessing pain in laboratory mice by monitoring gene expression changes 37 1.3. Pain models studied in this project 39 1.3.1. Surgical pain model: telemetric apparatus implantation 39 1.3.2. Inflammatory pain model: immunization with a BHV-1 virus 40 1.4. Microarray Technology 42 1.4.1. Competitive hybridization protocols 43 1.4.2. Affymetrix GeneChip® Mouse Exon 1.0 ST Array 45 1.5. Project description and relevancy 46 1.5.1. Rational of the project 46 1.5.2. Aims of the project 47 1.5.3. Relevancy of the project for animal welfare 48 2. Results 51 2.1. Low density microarray design 51 2.2. Whole Brain microarray experiments 52 2.3. Microarray experiments with specific regions of the central nervous system 58 2.3.1. Microarray results in the cortex 58 2.3.2. Microarray results in the brainstem 59 2.3.3. Microarray results in the cerebellum 61 2.3.4. Microarray results in the hippocampus 63 1 Table of contents 2.3.5. Microarray results in the spinal cord 65 2.4. Microarray protocol validation 66 2.4.1. Brainstem against cortex hybridizations 67 2.4.1.1. Brainstem against cortex hybridizations, raw signals comparisons 68 2.4.1.2. Brainstem against cortex hybridizations, normalized signals comp. 70 2.4.2. Hippocampus against brainstem hybridization 72 2.4.2.1. Hippocampus against brainstem hybridization, raw signals comp. 73 2.4.2.2. Hippocampus against brainstem hybridization, normalized signals 75 2.4.3. Hippocampus against cortex hybridization 78 2.4.3.1. Hippocampus against cortex hybridizations, raw signals comparisons 78 2.4.3.2. Hippocampus against cortex hybridizations, normalized signals comp. 82 2.5. Real-Time PCR measurements, telemetric apparatus pain model 84 2.6. Real-Time PCR measurements, inflammatory pain model 94 2.7. Whole Genome experiment in the spinal cord, telemetric apparatus 99 2.7.1. Whole gene regulations 99 2.7.2. Clustering analysis 102 2.7.2.1. Bi-clustering analysis of the downregulated genes 102 2.7.2.2. Bi-clustering analysis of the upregulated genes 105 2.7.3. Splice variant analysis 108 3. Discussion 112 3.1. Low density microarray 112 3.1.1. Whole brain microarray experiments 3.1.2. One-color model hybridization protocol validation 113 3.1.3. Microarray experiments on specific regions of the central nervous system 116 3.1.3.1. Microarray experiments on the brainstem 116 3.1.3.2. Microarray experiments on the cerebellum 117 3.1.3.3. Microarray experiments on the spinal cords 120 3.1.4. conclusions, low density microarray experiments 120 3.2. Real-Time PCR measurements on 27 candidate genes 121 3.3. Whole genome experiment in the spinal cord 122 4. Conclusions and Outlook 128 5. Material and methods 133 5.1. Low density microarray establishment 133 5.1.1. Gene selection and probes design 133 5.1.2. Spotting of oligos 133 5.2. Animal handling and treatments 134 5.2.1. Animal housing 134 2 Table of contents 5.2.2. Telemetric implantation 134 5.2.3. Inflammation model 135 5.3. Central nervous system dissection and tissue preservation 136 5.3.1. Brain dissection 136 5.3.2. Spinal cord dissection 136 5.3.3. Dorsal root ganglia dissection 137 5.4. RNA extraction 137 5.4.1. Whole brain RNA extraction 137 5.4.2. Parts of brain RNA extraction 137 5.4.3. Spinal cord RNA extraction 138 5.4.4. Dorsal root ganglia RNA extraction 138 5.5. aRNA amplification for the low density microarray hybridization 139 5.6. aRNA labeling 140 5.6.1. Whole brain and spinal cord aRNA labeling 140 5.6.2. Parts of brain RNA aRNA labelling 140 5.7. aRNA Hybridization 141 5.7.1. Whole brain aRNA hybridization 141 5.7.2. Spinal cord aRNA hybridization 142 5.7.3. Parts of brain aRNA hybridization 143 5.7.4. Slide scanning and data quantification 144 5.8. Low density microarray data analysis and normalization 145 5.8.1. Whole Brain hybridization data 145 5.8.2. Spinal cord and parts of brain data 145 5.8.3. Comparison between parts of brain hybridization data 146 5.9. Low-density microarray protocol validation 146 5.10. Quantitative Real Time PCR analysis 147 5.10.1. Reverse Transcription of total RNA 147 5.10.2. Q-RT PCR analysis of the differentially expressed genes in the low density microarray experiment 147 5.10.3. Quantitative Real-Time PCR analysis of 27 selected genes 149 5.10.4. Data interpretation 150 5.11. GeneChip® Mouse Exon 1.0 ST Arrays 151 5.11.1. cRNA preparation 151 5.11.2. Hybridization and data processing 151 5.11.3. Data analysis and normalization 152 5.11.4. Clustering analysis 152 6. Litterature 155 7. Acknowledgements 172 3 Table of contents 8. Supplementary data on CD-Rom 8.1. Appendix 1: List of genes spotted on the low-density microarray 1 8.2. Appendix 2: Design of the first microarray 7 8.3. Appendix 3: Design of the second microarray 8 8.4. Appendix 4: Design of the third microarray 9 8.5. Appendix 5: List of the filtered 170 genes with at least 3 downregulated exons in the spinal cord of the operated mice 10 8.6. Appendix 6: List of the filtered 25 genes with at least 3 downregulated exons in the spinal cord of the operated mice 15 8.7. Appendix 7: Top 1310 genes with splicing events in the spinal cord STD DV (Fold Change)>0.4 16 4 Table of contents 5 Curriculum Vitae Curriculum vitae Igor Asner Date of Birth : May 31st, 1980 Citizenship : Switzerland Marital status : Single, no children Professional Experience 01/ 2006 – Today PhD Student in Biology Institute for Laboratory Animal Science, University of Zurich Supervisor: Prof. K. Bürki and Dr. Paolo Cinelli Research Theme: Monitoring of nociception by measuring gene expression in the central nervous system of mice 05/ 2007 – 08/2008 Freelance scientific clinical trial collaborator BioPartners GmbH Supervisor: Dr. C. Savoy, CSO Function: Presentation and figure preparation for Phase III clinical trial reports on the sustained-release recombinant Human Growth Hormone Experience with ICH/GCP guidelines and clinical trial operations 06/ 2006 – Today Tutor, Introductory Course in Laboratory Animal Science (Module 1) Institute for Laboratory Animal Science, University of Zurich Supervisor: Dr. H-P. Käsermann 6 Curriculum Vitae 10/ 2004 – 07/ 2005 Secondary School Biology and Mathematics teacher Collège des Coudriers, Geneva 09/ 2004 – 12/ 2004 Collaborator on the “Quality of Life Assessment for Elderly People with Dementia” research project Psychology Faculty, University of Geneva Supervisor; Prof. N. Von Steinbüchel, University of Geneva Function: Interviews of patients living with dementia 08/ 2003 – 10/ 2003 Freelance journalist 09/ 2002 – 04/ 2004 Diploma Student in Biology Neuroscience Institute, Swiss Federal Institute of Technology in Lausanne Supervisor: Prof. P. Aebischer and Prof. W. Pralong Research Theme: Establishment of a cell line for the in-vitro study of the GDNF receptor Education 01/ 2006 – Today PhD in Biology University of Zurich, Switzerland 09/ 2002 − 04/ 2004 Diploma in Biology University of Geneva and Swiss Federal Institute of Technology in Lausanne, Switzerland 09/ 1998 – 06/ 2002 Licence of Science in Biology University of Geneva, Switzerland 7 Curriculum Vitae 09/ 1995 – 06/ 1998 High school education International School of Ferney-Voltaire, France Scientific Baccalauréat, specialisation in Biology KMK German Language Diploma, Advanced Level General Certificate of Secondary Education: English, English Literature and Mathematics Languages French: Mother tongue English: Excellent German: Excellent Croatian: Excellent 8 Publications Publications “Genetic vasectomy - Overexpression of Prm1-EGFP fusion protein in elongating spermatids causes dominant male sterility in mice” Sabine Haueter, Miyuri Kawasumi, Igor Asner, Urszula Brykczynska, Paolo Cinelli, Stefan Moisyadi, Kurt Bürki, Antoine H.F.M.