Molecular Genetic Analysis of Pain and Stress in the Laboratory Mouse

1. Molecular Genetic Analysis • of Pain and Stress in the • Laboratory Mouse

2. Goal 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.

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

4. 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. Definition of Pain Painis 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. Cerebral Cortex Thalamus Nociceptors Touch/Pressure receptors From Brain To Brain Sensory Nerves Dorsal Horn Muscle Fibers Motor Nerve Fibers Pain Perception

7. 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. Therefore, it is plausible to suppose that mice can experience pain 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.

9. Pain Perception - Defensive Behavior - Scream - Disturbances in the movements - Retreat into a safe and quiet place

10. Objective Measurement of Pain • Analysis of physiological parameters • Heartrate • Body temperature - Behavior Analysis Activity - Analysis of biochemical values Hormones in the blood

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

14. The pain-related factors are produced in the cells and afterwards transported around the body. examples: Glands Spinal cord cells Brain cells Neurons

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

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

17. The information needed for the production of a pain-factor is contained in a so called Gene Gene 1 Gene 2

18. Genes contain the information for the synthesis of pain-factors (Proteins) DNA Trascription mRNA Cell Translation Protein

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

20. 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 Purification Labeling Maus w/o Pain

23. Complementary nucleic acids can bind to each other DNA A DNA B DNA A DNA B

24. The labeled DNAs can be distributed on the Microarray

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

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

27. = Mouse without pain = Mouse with Pain Analysis Colour 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

28. The DNA-Microarray Technology: Summary

29. 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. The DNA-Microarray Technology allows: • 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

31. The DNA-Microarray Technology allows the simultaneous analysis of tausend of genes