1. Institute of Laboratory Animal Science University of Zürich Molecular Genetic Analysis of Pain and Stress in the Laboratory Mouse

2. Institute of Laboratory Animal Science University of Zürich The goal of this project is the design of a DNA-microarray for the detection and quantification of pain and Stress in the laboratory mouse. Goal

3. Institute of Laboratory Animal Science University of Zürich Measurements of physiological parameters (Heartrate, body temperature, activity) Margarete Arras Paolo Cinelli Behavioural Analysis Margarete Arras Hanno Würbel DNA-Microarrays (Genetic Analysis)) Paolo Cinelli Stress and Pain detection in the Mouse

4. Institute of Laboratory Animal Science University of Zürich Applications - Identification of pain/stress influence during the analysis of transgenic animals (Overexpression, Knockout). E.g. Behavioral tests... - Analysis and refinement of anaesthesia (which kind of anaesthesia is necessary for a defined operation) - Clear definition of pain during animal experimentation - Optimization of the housing conditions for the animals

5. Institute of Laboratory Animal Science University of Zürich Definition of Pain Pain is a phenomenon, that contains both a physical and a psycological component. It is a symptom of many diseases and is very important for the perception of physical and mental dysfunctions of the body. Acute Pain is always an Alarm indicating a dysfunction in the body and is an early warning system to protect the body from further damages. Pain is from this point of view the first step in the process leading to recovery.

6. Institute of Laboratory Animal Science University of Zürich Pain Perception Nociceptors Touch/Pressure receptors Cerebral Cortex Thalamus Motor Nerve Fibers Muscle Fibers From Brain To Brain Sensory Nerves Dorsal Horn

7. Institute of Laboratory Animal Science University of Zürich The definition of pain in animals is very unprecise for different reasons: - pain is a subjective condition - and for this reason, strictly speaking, is applicable only for humans

8. Institute of Laboratory Animal Science University of Zürich Can Mice feel Pain? - The nervous system of mice is very similar to the one of humans. - Mice and humans have similar physiological processes. - Mice and humans behave under pain conditions in a very similar way. Therefore, it is plausible to suppose that mice can experience pain

9. Institute of Laboratory Animal Science University of Zürich Pain Perception - Defensive Behavior - Scream - Disturbances in the movements - Retreat into a safe and quiet place

10. Institute of Laboratory Animal Science University of Zürich Objective Measurement of Pain - Analysis of physiological parameters Heartrate Body temperature - Analysis of biochemical values Hormones in the blood - Behavior Analysis Activity

11. Institute of Laboratory Animal Science University of Zürich Physiological values and activity can be measured directly in the animals by a telemetric system The transmitters are implanted into the mice and allow the measurement of Heartrate, Body Temperature an Activity.

12. Institute of Laboratory Animal Science University of Zürich Analysis of Biochemical Values The production and regulation of factors related to pain and stress is very complicated and involves different regions of the body. An exact biochemical analysis is only possible by simultaneous measurement of many different factors.

13. Pain signals STIMULATING Glutamate Substance P Neurokinin A Other neuropeptides Prostaglandines INHIBITING  Endorphine Noradrenaline Dynorphin Endomorphine GABA STIMULATING Prostaglandines Bradykinin Histamine Leukotrien Growth factors Substance P other neuropeptides INHIBITING Anandamides  Endorphine Pain Signals Inhibiting The Biochemistry of Pain

14. Institute of Laboratory Animal Science University of Zürich The pain-related factors are produced in the cells and afterwards transported around the body. examples: Glands Spinal cord cells Brain cells Neurons

15. Institute of Laboratory Animal Science University of Zürich Nervenzelle Nervous system cells (Neurons) are responsible for the transport of the pain/stress signals Axon Cell body Neuron Cell body Axon Neurotransmitter Synapse Receptor

16. Cell Cell body Chromosome DNA The nucleus of the cells contains the Information necessary for the production of pain-related Substances. This information Is the DNA.

17. Institute of Laboratory Animal Science University of Zürich The information needed for the production of a pain-factor is contained in a so called Gene Gene 2 Gene 1

18. Institute of Laboratory Animal Science University of Zürich DNA mRNA Protein Trascription Translation Genes contain the information for the synthesis of pain-factors (Proteins) Cell

19. Institute of Laboratory Animal Science University of Zürich The analysis of the genome of the mouse allows the identification of the genes involved in the pain/stress processes mRNA In order to perform this analysis it is necessary to isolate the nucleic acids ( ) from the cells.

20. Institute of Laboratory Animal Science University of Zürich The DNA-Microarray Technology is a very powerfull technique that allows a fast analysis of hundreds of Pain related genes.

21. The DNA-Microarray Technology The nucleic acids (genes), that are involved in prodution of pain-related factors are spotted on a carrier (membrane, glass,...) DNA solution Transfer to Glass DNA-Microarray

22. The nucleic acids purified from a mouse with pain and a mouse without pain are labelled with specific fluorescent substances. Maus with Pain Maus w/o Pain PurificationLabeling

23. Institute of Laboratory Animal Science University of Zürich Complementary nucleic acids can bind to each other DNA A DNA B DNA A DNA B

24. Institute of Laboratory Animal Science University of Zürich The labeled DNAs can be distributed on the Microarray

25. The DNA-Probes on the microarray can bind the fluorescence-labeled nucleic acids.

26. The detection can be performed by a laser Laser 1 Red Laser 2 Green

27. Colour Analysis + = Gene-expression is not changed during Pain/Stress Increased gene expression during Pain/Stress Gene expression is reduced during Pain/Stress Analysis of the DNA-Microarrays = Mouse with Pain= Mouse without pain

28. The DNA-Microarray Technology: Summary

29. Institute of Laboratory Animal Science University of Zürich Die DNA-Microarray Technology offers one of the more reliable analysis methods for Pain/Stress detection Microarrays allow the simultaneous quantification of many different factors involved in pain/stress production and management (e.g. Neurotransmitters, receptors, Hormones,...)

30. Institute of Laboratory Animal Science University of Zürich - Identification of pain/stress influence during the analysis of transgenic animals. - Analysis and refinement of anaesthesia - Clear definition of pain during animal experimentation - Optimization of the housing conditions for the animals The DNA-Microarray Technology allows:

31. Institute of Laboratory Animal Science University of Zürich The DNA-Microarray Technology allows the simultaneous analysis of tausend of genes