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Molecular Genetic Analysis of Pain and Stress in the Laboratory Mouse Institute of Laboratory Animal Science University of Zürich 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. Institute of Laboratory Animal Science University of Zürich Stress and Pain detection in the Mouse Measurements of physiological parameters (Heartrate, body temperature, activity) Margarete Arras Paolo Cinelli Behavioural Analysis DNA-Microarrays (Genetic Analysis)) Paolo Cinelli Margarete Arras Hanno Würbel 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 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. Institute of Laboratory Animal Science University of Zürich Pain Perception Cerebral Cortex Thalamus Nociceptors Touch/Pressure receptors Sensory Nerves From To Brain Brain Dorsal Horn Muscle Fibers Motor Nerve Fibers 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 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 Institute of Laboratory Animal Science University of Zürich Pain Perception - Defensive Behavior - Scream - Disturbances in the movements - Retreat into a safe and quiet place Institute of Laboratory Animal Science University of Zürich Objective Measurement of Pain - Analysis of physiological parameters Heartrate Body temperature - Behavior Analysis Activity - Analysis of biochemical values Hormones in the blood 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. 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. Institute of Laboratory Animal Science University of Zürich The Biochemistry of Pain INHIBITING b-Endorphine Noradrenaline Dynorphin Endomorphine GABA Pain signals Inhibiting INHIBITING Anandamides b-Endorphine STIMULATING Glutamate Substance P Neurokinin A Other neuropeptides Prostaglandines Pain Signals STIMULATING Prostaglandines Bradykinin Histamine Leukotrien Growth factors Substance P other neuropeptides The pain-related factors are produced in the cells and afterwards transported around the body. examples: Glands Spinal cord cells Brain cells Institute of Laboratory Animal Science Neurons University of Zürich Nervous system cells (Neurons) are responsible for the transport of the pain/stress signals Neuron Receptor Axon Cell body Nervenzelle Cell body Axon Synapse Neurotransmitter Institute of Laboratory Animal Science University of Zürich Chromosome The nucleus of the cells contains the Information necessary for the production of pain-related Substances. This information Is the DNA. Cell body Cell DNA The information needed for the production of a pain-factor is contained in a so called Gene 1 Gene 2 Gene Institute of Laboratory Animal Science University of Zürich Genes contain the information for the synthesis of pain-factors (Proteins) DNA Trascription mRNA Cell Translation Protein 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 In order to perform this analysis it is necessary to isolate the nucleic acids ( mRNA) from the cells. 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. Institute of Laboratory Animal Science University of Zürich 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 The nucleic acids purified from a mouse with pain and a mouse without pain are labelled with specific fluorescent substances. Maus with Pain Purification Maus w/o Pain Labeling Complementary nucleic acids can bind to each other DNA A DNA B DNA A DNA B Institute of Laboratory Animal Science University of Zürich The labeled DNAs can be distributed on the Microarray Institute of Laboratory Animal Science University of Zürich The DNA-Probes on the microarray can bind the fluorescence-labeled nucleic acids. The detection can be performed by a laser Laser 1 Red Laser 2 Green Analysis of the DNA-Microarrays = Mouse without pain Colour = + = Mouse with Pain Analysis Gene-expression is not changed during Pain/Stress Increased gene expression during Pain/Stress Gene expression is reduced during Pain/Stress The DNA-Microarray Technology: Summary 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, ...) Institute of Laboratory Animal Science University of Zürich 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 Institute of Laboratory Animal Science University of Zürich The DNA-Microarray Technology allows the simultaneous analysis of tausend of genes Institute of Laboratory Animal Science University of Zürich