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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