Institute of Laboratory Animal Science University of Zürich

Molecular Genetic Analysis

of Pain and Stress in the

Laboratory Mouse

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

Institute of Laboratory Animal Science University of Zürich

Measurements of physiological parameters (Heartrate, body temperature, activity)

Margarete ArrasPaolo Cinelli

Behavioural Analysis

Margarete ArrasHanno Würbel

DNA-Microarrays(Genetic Analysis))

Paolo Cinelli

Stress and Pain detection in the Mouse

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

Nociceptors

Touch/Pressurereceptors

Cerebral Cortex Thalamus

Motor NerveFibers

MuscleFibers

FromBrain

To Brain

Sensory Nerves

Dorsal Horn

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 canexperience 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 parametersHeartrate

Body temperature

- Analysis of biochemical valuesHormones in the blood

- Behavior Analysis Activity

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.

Pain signals

STIMULATINGGlutamateSubstance PNeurokinin AOther neuropeptidesProstaglandines

INHIBITINGEndorphineNoradrenalineDynorphinEndomorphineGABA

STIMULATINGProstaglandinesBradykininHistamineLeukotrienGrowth factorsSubstance Pother neuropeptides

INHIBITINGAnandamidesEndorphine

P

ain

Sig

na

ls

Inh

ibit

ing

The Biochemistry of Pain

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 cellsBrain cells

Neurons

Institute of Laboratory Animal Science University of Zürich

Nervenzelle

Nervous system cells (Neurons) are responsiblefor the transport of the pain/stress signals

Axon

Cell body

Neuron

Cell body

Axon

Neurotransmitter

Synapse

Receptor

Cell

Cell bodyChromosome

DNA

The nucleus of the cells containsthe Informationnecessary forthe production of pain-relatedSubstances. This informationIs the DNA.

Institute of Laboratory Animal Science University of Zürich

The information needed for

the production of a pain-factoris contained in

a so calledGene

Gene 2

Gene 1

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

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.

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.

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

Maus w/o Pain

Purification Labeling

Institute of Laboratory Animal Science University of Zürich

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

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

The detection can be performed by a laser

Laser 1Red

Laser 2Green

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

The DNA-Microarray Technology: Summary

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, ...)

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:

Institute of Laboratory Animal Science University of Zürich

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