Department of Pathophysiology1
Principles and ethics of experimental use of
laboratory animals
Ing. Hana Holcová Polanská, Ph.D
Mgr. Lucie Válková, Ph.D.
Department of Pathophysiology2
Use of live systems
The idea of using animals in experiments
Claude Bernard (1813-1878) – physician
̶ 1867 publication l'Introduction à l'étude de la médecine
expérimentale (Introduction to the study of experimental medicine)
̶ „ The principles of experimental medicine are the principles of all experimental sciences.“
̶ laboratory mouse (Mus musculus)
̶ laboratory rat (Rattus norvegicus)
̶ laboratory rabbit (Oryctolagus cuniculus)
̶ golden hamster (Mesocricetus auratus)
̶ guinea pig (Cavia apperea)
̶ pig (Sus scrofa)
̶ African clawed frog (Xenopus laevis)
̶ fish - e.g. striped danio (Danio rerio)
Department of Pathophysiology3
The most commonly used exp. animals
Figures from: https://www.biolib.cz/
& https://www.velaz.cz/
Department of Pathophysiology4
Animal production systems for experiments
1. Open
̶ Free entrance of animals, persons and material, barrier-free
2. Closed
a) Barrier housing
• animals separated from the outside environment by a barrier and hygiene
loop, not separated from humans
• SPF animals
b) Isolator
• animals confined in isolators, which separates them from the outside
environment and humans
• axenic, gnotobiotic animals
Department of Pathophysiology5
According to housing method
̶ Conventional
̶ common facilities, open breeding and housing system, control of pathogens
transmissible to humans
̶ SPF
̶ closed breeding and housing system, Specific Patogen Free animals
̶ Axenic
̶ closed breeding and housing system
̶ NO micro-organisms
̶ Gnotobiotick
̶ “gnotos” (known) and “bios” (life)
̶ closed breeding and housing system
̶ axenic animal + defined microorganism (mono-, di-, poly-xenic)
Department of Pathophysiology6
Barrier housing - scheme
MATERIAL
INPUT FOR
STERILIZATION
CLEAN CORRIDOR
AUTOCLAVE
CLEAN ROOM
SOILED ROOM
ANIMAL
ROOM
ANIMAL
ROOM
ANIMAL
ROOM
ANIMAL
ROOM
ENTRANCE
FROM THE
HYGIENE LOOP
SOILED CORRIDOR
Department of Pathophysiology7
Isolators
University of Calgary Ohio State University
Department of Pathophysiology8
Animal models – a genetics perspective
Inbred
̶ colonies formed by at least 20 generations of brother-sister breeding that can be traced back to a single
founding pair
Outbred
̶ crossbreeding colonies that have been geographically separated (different breeding facility) until they
reach the same range of genetic variation
Hybrid
̶ by crossbreeding two inbred strains. This cross produces hybrid animals that are more resistant to
disease, have higher survival rates under stress, live longer and have larger litters than the parent
strains
Trangen
̶ used in almost every therapeutic and disease research area
̶ DNA modification (knock-out functional gene, knock-in a non-functional/muted gene, inserting a piece of
foreign DNA, …)
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Animal models – immune system perspective
Immunocompetent
̶ no changes in the immune system
Immunodeficient
̶ animals with different levels of immunodeficiency and phenotypic characteristics
Humanised animal models
̶ induced human immune system of various levels
Department of Pathophysiology10
Using animals in the experiments
The 3Rs principle
̶ 1959 Russel a Burche
› Reduction
› Replacement
› Refinement
More information about the history of the 3Rs principles at:
https://norecopa.no/media/2ltdq3ht/the-three-rs_120324.pdf
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Reduction
̶ any procedure that results in the same amount of information
being obtained using fewer animals or maximising the information
obtained from a single animal
̶ appropriate experimental design and adequate analysis of the
resulting data, with due consideration of statistical principles, can
increase the precision of the results and equally allow the use of
fewer animals to develop the data
̶ a small pilot study will indicate whether or not it is appropriate to
proceed to the main experiment
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Replacement
̶ any experimental method that does not require the use of a
whole, live animal
̶ some of these methods only partially replace
̶ humane killing of animals to obtain cells, tissues or organs for
subsequent studies in vitro
Other methods substitute fully, as they do not require any biological
material obtained from a fully developed vertebrate.
Department of Pathophysiology13
Animal experimentation alternatives
̶ Consistent mining of existing experimental data
̶ Models, computer programs
̶ Physico-chemical techniques
̶ Invertebrates, plants, microorganisms (lower organisms)
̶ Early developmental stages - bird egg germarium, embryos
̶ Human volunteers, epidemiological data
̶ Cell, tissue and organ cultures (relative surrogates - require
freshly obtained cells or tissue)
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Refinement
̶ modifying any procedure that interferes with the life of a
laboratory animal from birth to death, so that the pain and
suffering experienced by the animal is minimised and its standard
of living is raised
̶ staff should be well trained and skilled in the handling of the
species of animals used
̶ anaesthesia and analgesia should be used wherever appropriate
and possible
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Regulation of use of experimental animals
̶ Animal Protection Act No. 246/1992 Coll. (which is the
implementation of Directive 2010/63/EU of the European
Parliament and of the Council of 22 September 2010 on the
protection of animals used for scientific purposes)
̶ Central Commission for the Protection of Animals (ÚKOZ)
̶ Expert Committee for Ensuring the Welfare of Experimental
Animals
Ústav patologické fyziologie16
Expert Committee for Ensuring the Welfare
of Experimental Animals
̶ at every supplier, breeder and user facility
̶ provides consultancy on the welfare of experimental
animals, the 3Rs, ...
̶ establish, monitor and review internal operating procedures
̶ monitor and control the progress and results of experimental
projects
Department of Pathophysiology17
Central Commission for the Protection of
Animals (ÚKOZ)
̶ protection of livestock animals
̶ protection of pet animals
̶ protection of wild animals
̶ protection of experimental animals
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Animal Protection Act No. 246/1992 Coll.
§ 3 Terms
a) an animal is any living vertebrate except a human being, but not a fetus or embryo,
…
j) experimental animal
1. a living vertebrate animal which is or is to be used for experiments, other than a
human being, including self-feeding larval forms and mammalian fetuses from
the last third of their normal development, or
2. a live cephalopod which is or is to be used for experiments;
an experimental animal shall also be considered to be an animal which is at an
earlier stage of development than that referred to in point 1, if the animal is to be
allowed to live beyond that stage of development and, as a result of the
experiments carried out, is likely to suffer pain, suffering, distress or permanent
harm when it reaches that stage of development,
Department of Pathophysiology19
Animal Protection Act No. 246/1992 Coll.
§ 18 Purposes of the experiments
(1)Experiments may be carried out for these purposes only:
f) higher education or training to acquire, maintain or improve
professional skills
Department of Pathophysiology20
Probator laparotomy
- animal model
Ing. Hana Holcová Polanská, Ph.D.
Mgr. Lucie Válková, Ph.D.
Department of Pathophysiology21
Probator laparotomy
Department of Pathophysiology22
Hands-on
1) Animal in full surgical tolerance
2) Place the animal in the supine position it on the operating table
3) Laparotomy
4) Observation of the organs of the abdominal cavity
5) Suture of the muscle layer
6) Suture of the skin layer
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Hands-on
1) Animal in full surgical tolerance
= under deep anaesthesia
- No further addition of anesthesia
will be necessary
Department of Pathophysiology24
Hands-on
2) Place the animal in the supine
position on the operating table
- Use a rubber band to fix the
thoracic and pelvic limbs
- To fix the head, fasten the rubber
band behind the upper teeth
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Hands-on
3) Laparotomy
- Do a laparotomy in abdominal region.
1. Open the skin layer
2. Open the muscle layer.
- The incision will run from the processus
xiphoideu almost to the symphysis.
Be careful not to cut into the thorax!
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Hands-on
4) Observation of the organs of the
abdominal cavity
̶ caecum vs. Stomach
̶ kidneys (both) + vena renalis
̶ aorta abdominalis + vena cava caudalis
̶ urinary bladder, …
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Hands-on
5) Stitching of the muscle layer
- Use of the continuation stitch
- Round needle on the cross-section
- Distance between stitches about 5-7 mm
- Tighten the stitches properly so that the edges of
the tissues fit tightly together
- Do not over-tighten the sutures to avoid tissue
ischemia (reducing healing)
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Hands-on
6) Stitching of the skin layer
- Use of individual stitches
- Triangular needle on the cross-section
- Distance between stitches about 5-7 mm
- Shorten the ends of the stitches to
approx. 5 mm
- Tighten the stitches properly so that the edges of the tissues fit
tightly together
- Do not over-tighten the sutures to avoid tissue ischemia
(reducing healing)
Department of Pathophysiology29
Notes
If you become unwell (for any reason), do not worry and report
this to the teacher immediately (to prevent an accident).
If the animal begins to move on its own at any time (showing
signs of anesthesia wearing off; eg. move its tail, or raise its
paw/s), report this to the teacher immediately.
Department of Pathophysiology30
Eating, drinking and
smoking are forbidden in
the laboratory areas
Department of Pathophysiology31
It is forbidden to take
ANY records from
the laboratory areas