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DO DIFFERENT CAGING SYSTEMS AFFECT THE OUTCOME OF ... · , Balafas E. 1, Alexakos P. 1,...
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DO DIFFERENT CAGING SYSTEMS AFFECT THE OUTCOME OF BEHAVIORAL TESTS IN C57BL/6J MALE
MICE?
Moltsanidou E.1, Polissidis A.1, Dalla C.2, Balafas E.1,
Alexakos P.1, Kostomitsopoulos N.1
1 Centre of Clinical, Experimental Surgery and Translational Research,
Biomedical Research Foundation of the Academy of Athens, Greece2 Department of Pharmacology, Medical School,
National and Kapodistrian University of Athens, Greece
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INTRODUCTION
Housing can affect the health and well-being of laboratory mice.
→ May affect the outcome and the reliability of the experimentalresults.
The purpose of this study was to assess the role of the cagingsystem in the exploratory and anxiety-related behavior of mice.
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Laboratory mice housing systemsLaboratory mice cages must: be constructed of safe, durable materials;
be kept clean;
be maintained in good repair;
be secure and escape-proof;
protect mice from climatic extremes;
not cause injury to mice;
be large enough for the number of animals held; and
be compatible with the behavioral needs of the mice.
provide the availability to observe the animals readily.
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Open Top Cages – OTCs Free exchange of air;
The intracage environment is influenced • by the animal room's environment (macroenvironment);• the frequency of bedding changes, and the • housing density.
Allows cage-to-cage and room-to-cage transmission of airborne pathogens;
Does not protect staff from exposure to animal-related aeroallergens.
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Individually Ventilated Cages – IVCs
Provide a significant barrier to thespread of infectious agents;
Protect staff from aeroallergens andzoonotic agents (quarantine);
Improve the air quality in the cage(high-efficiency particulate filtration ofthe incoming air-HEPA);
Reduce the frequency of cage andbedding changes;
Negative or positive intracage pressurecan be defined;
Ventilation rates may vary from 25 to120 air changes per hour (ACH).
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Motor Free Ventilated Cages – MFVCs
Connected directly to the exhaust of the room's heating, ventilation, and air conditioning (HVAC) unit;
Protect the animals against pathogens; Protect the staff against animal-related
aeroallergens; Operate only under negative pressure;
Ventilation rates (20–25 ACH) can't be modified.
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MATERIALS AND METHODS
Thirty six 25-days-old C57BL/6, male mice were randomly divided into threeequal groups (n = 12):
Group 1: OTCs (10-15 ACH)
Group 2:IVCs
(70 ACH, positive
pressure)
Group 3:MFVCs
(25 ACH, negative pressure)
Mice were housed for two months prior to behavioral testing.
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Elevated Plus Maze test – EPM
The following parameters were evaluated:(i) time spent in open and closed arms (seconds), (ii) number of entries into the open and closed arms, and (iii) locomotor activity measured as the total distance travelled (cm). Anxiety-like behavior of each mouse was determined based on the number of entries and/or the time spent in the open arms.
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Elevated Plus Maze test
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Open Field test
The following parameters were evaluated: (i) locomotor activity, as measured by the total distance travelled (cm) and (ii) anxiety-related behavior, as measured by the total amount of time a
mouse spent in the central area of the arena (20 cm× 20 cm).
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Open Field test
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Three-Chamber Sociability and Social Novelty test
Rodents normally prefer to spend more time with another rodent(sociability) and will investigate a novel intruder more so than a familiarone (social novelty).
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Three-Chamber Sociability and Social Novelty test
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Three-Chamber Sociability and Social Novelty Test
The following parameters were evaluated:
(i) Time spent in each chamber (s).
(ii) The number of entrances in each chamber during the second phase.
(iii) The number of entrances in each chamber during the third phase.
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RESULTS
All statistical analyses were conducted using one-way of GraphPad PrismV6. Data are expressed as mean ± standard error of the mean (SEM) andstatistical significance was set at 5%.
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Elevated Plus Maze test
M F V C O T C IV C0
2 0
4 0
6 0
8 0
1 0 0
*# #
F(2,31)=5.833, p=0.0071
Housing Groups
% n
umbe
r of e
ntrie
s in
to th
e op
en a
rms
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Elevated Plus Maze test
M F V C O T C IV C0
1 0
2 0
3 0
4 0
5 0
*# #
F=5.833, p=0.0071
% n
umbe
r of e
ntrie
s in
to th
e cl
osed
arm
s
Housing Groups
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Open Field test
M F V C O T C IV C0
1 0 0 0
2 0 0 0
3 0 0 0 **#
F(2, 16) = 4.808, p= 0.0232
Housing Groups
Tota
l dis
tanc
e tr
avel
led
(cm
)
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Open Field test
M F V C O T C IV C0
2 0
4 0
6 0
8 0 *# #
F(2,16) = 5.006, p= 0.0205
Housing Groups
Tim
e sp
ent i
n th
e ce
nter
of t
he
aren
a (s
).
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Three-Chamber Sociability and Social Novelty test
MF V C
O T CIV
C0
1 0 0
2 0 0
3 0 0
F(2, 32)=8.016, p=0.0079
Tim
e (s
)
Housing Groups
Sociability test
Stranger 1Empty chamber
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Three-Chamber Sociability and Social Novelty test
S o c ia l n o v e lty te s t
H o u s in g G ro u p s
Tim
e (
s)
MF V C
OT C
IVC
0
1 0 0
2 0 0
3 0 0
4 0 0S T R A N G E R 1
S T R A N G E R 2
F(2, 32)= 8.281, p= 0.0071
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DISCUSSION (I)
o Results from the Elevated Plus Maze test revealed that mice housed in the MFVCs showed increased exploratory and less anxiety-like behavior.
→ Tend to show higher locomotor activity and spend more time in the center of the arena compared to OTC and IVC housed mice.
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DISCUSSION (II)
o Results from the Open Field test revealed that mice housed in the MFVCs showed increased exploratory and less anxiety-like behavior.
→ Tend to spend more time in the central area and to express higher locomotor activity compared to OTC and IVC caged mice.
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DISCUSSION (III)
o The Three Chamber Sociability and Social Novelty test showed that mice of all three caging systems equally preferred to spend more time with another mouse (sociability) and to investigate a novel mouse more than a familiar one (social novelty).
→ No significant difference was observed between the mice of the three groups MFVC, OTC and IVC.
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CONCLUSIONS
o It is concluded that different caging systems may influence theexploratory and anxiety-like behavior of laboratory mice.
Differences related to: The design of the cages. Air changes per hour. Noise. Pherormones.
o It is essential to take into account the housing conditions when designingand performing experimental protocols as well as when reporting,analyzing, and systematically reviewing the results of behavioral testingin mice.
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LITERATURE:
1. Baumans, V., & Van Loo, P. L. P. (2013). How to improve housing conditions of laboratory animals: The possibilities of environmental refinement. Veterinary Journal. https://doi.org/10.1016/j.tvjl.2012.09.023
2. Baumans, V. (2010). The Laboratory Mouse. In The UFAW Handbook on the Care and Management of Laboratory and Other Research Animals: Eighth Edition. https://doi.org/10.1002/9781444318777.ch21
3. Russell, WMS & Burch, RL (1959). The principles of humane experimental Technique, London:Methuen, Facsimile edition, p:238, Potters Bar, Herts, UK:UFAW, 1992
4. Van De Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., & Baumans, V. (1997). Nesting material as environmental enrichment has no adverse effects on behavior and physiology of laboratory mice. Physiology and Behavior. https://doi.org/10.1016/S0031-9384(97)00232-1
5. Polissidis, A., Nikita, M., Alexakos, P., Stasinopoulou, M., Kakazanis, Z., & Kostomitsopoulos, N. (2017). Physiology & Behavior Assessing the exploratory and anxiety-related behaviors of mice . Do di ff erent caging systems a ff ect the outcome of behavioral tests ?, 177(April), 68–73. https://doi.org/10.1016/j.physbeh.2017.04.009
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Thank you for your attention!