institute of laboratory animal science university of zürich molecular genetic analysis of pain and...
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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
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
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: