Critical care of sub-lethal irradiated transgenic mice

Critical care of sub-lethal irradiated transgenic mice using a complete soft food formula DietGel76A™
Ovidiu I. Jumanca, DVM, Cert. LAM, Jay Palmer, BSc
IRCM - Institut de recherches cliniques de Montréal, Clear H2O, Portland, MA
ABSTRACT
RESULTS
The objective of this research is to determine whether the administration of a complete soft food formula to sub-lethal irradiated
animals from three different transgenic mice strains over a period of 21 consecutive days, will have a significant impact on the
clinical signs, and the general survival rate of the animals. Our hypothesis was that using DietGel76A™, along with appropriate
care of animals, would be significantly beneficial to experimental animals after a total body irradiation for allogeneic bone marrow
transplantation.
For this experiment three different strains of mice were used: C57BL/6, (JAX stock 000664), NRG (JAX stock 007799) immunodeficient mice model and CD45.1 (JAX stock 002014) - strain most commonly used for the transplant of bone marrow
cells. In our experiment, no bone marrow transplants have been performed; therefore the reconstitution of hematopoietic system
was done exclusively by self-regeneration.
Our hypothesis was that using DietGel76A™ along with antibiotic treatment, strict handling and manipulation procedures, the
general mortality rate, as well as the onset of clinical signs between the treated animals and the control animals would be
significantly lower.
This hypothesis was clearly confirmed for the C57BL/6 strain. The treatment with DietGel76A™ had a net positive impact on the
recovery of more irradiation sensitive strains (CD45.1, and mostly NRG). However, in our opinion, mice belonging to more
sensitive strains should be on DietGel76A™ for a longer period of time (7 days pre-irradiation to 42 days post irradiation).
Mice from all groups pretreated with PBS and antibiotics displayed a survival rate of near to 100% over a 30 day period
following irradiation. Following the irradiation of the three strains used in this experiment, we discovered that the most
sensitive strain was the NRG (BW difference=1.95, R2=0.7565,  =24.62), followed by the CD45 (BW difference =2.85,
R2=0.7822,  =20.52) and the C57 (BW difference=2.10, R2=0.7989,  =19.80). Irradiated C57 mice supplemented
with DietGel had a significant BW improvement in comparison with the non-treated cohort (SS=19.31 ±3.34) (p
<0.05), see fig 2b). and fig 2h). Following irradiation, during the 21 days of the DietGel 76 A treatment, there is a clear
ascending trend of the BW for the treated group (IDG), versus non treated (INDG) group. The DietGel was withdrawn
after Day 22 of the study, which corresponds to a slight loss of the BW recovery trend in the treated group (fig 2i).
60
27.50
3.5
3
50
2.5
40
2
1.5
Mortality
20
b)
Survival
0
C57
Mortality
CD45.1 mice
NRG
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
C57BL6 mice
NRG mice
R² = 0.7989
R² = 0.7822
22.50
C57
d)
e)
17.50
f)
17.50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
22
21.5
3.50
26.00
3.00
25.50
2.50
25.00
24.50
IDG
24.00
INDG
Linear (IDG)
23.50
Linear (INDG)
23.00
22.50
20.5
20
2.10
IDG
1.95
2.00
19.5
1.50
19
1.00
18.5
0.50
18
0.00
22.00
g)
21
Variation in BW average of irradiated group animals after
40 days of treatment
2.85
h)
21.50
CD45
C57
CD45
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
C57
NRG
NRG
i)
INDG
17.5
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Irradiated with DietGel Vs Irradiated without DietGel treatment
*IDG= Irradiated and treated with DietGel
*INDG= Irradiated without DietGel treatment
Figure 2. Graphics and charts:
Upon reception of the animals at the IRCM, the randomization, group assignment and pre-irradiation body weight measurements have been
performed (Day 3) prior to irradiation. From Day 3 prior irradiation, the animals from SD groups ( sub-lethal+DietGel) received one can of DietGel
76A in cage, for acclimatization. On the same day, all the animals started receiving an antibiotic treatment in water.
On irradiation day, all the animals (except controls) were sub-lethally irradiated using Gamma-radiation produced using an irradiator which source is
constituted by Cesium-137. Doses of irradiation were based on the time of exposure calculated at the time of the experiment (conversion
1Gy/min=100 Rads/min, source delivering is 120.84 Rads/min). The amount of time spent inside the irradiator has been calculated depending on the
dose and radioisotope decay charts. Irradiation doses were sub-lethal. Each animal received 4Gy of gamma -irradiation, equivalent to 3min 15sec of
exposure (decay rate: 123.72 cGy/min).
Animal care and procedures, antibiotics and DietGel administration
Immediately after irradiation, all the animals were injected with 1 mL of sterile isotonic solution subcutaneously (Physiological Saline (0.9%), CDMV,
product # 1399, St.-Hyacinthe (QC), Canada). The injection was repeated after 24h.From Days 1 to Day 21 post irradiation, the following
observations were made: daily animal check, mortality check, clinical signs check, endpoints scores. Special scoring sheets (Fig. 1) were designed
to monitor the clinical signs and endpoints score. A decisional tree was designed to evaluate and take action according to the endpoints (Fig 2.).
Mice
C57BL/6J
stock 000664
Mice NRG
stock 007799
Mice
CD45.1 stock
002014
TOTAL
Linear (C57)
17.50
26.50
Irradiation procedures:
Strain
Linear (CD45)
Linear (NRG)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Experimental Design
Female mice, 4-6 weeks old, from The Jackson Laboratories (Bar Harbor, Maine 04609 USA) were used. For this experiment three different strains
of mice have been used:
o
C57BL6 (JAX stock 000664 - C57BL/6J ) - classical model used by many institutions, probably the most known Tg strain of mice.
o
NRG (JAX stock 007799 - NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) - immunodeficient mice commonly used for cell or tissue transplant
studies.
o
CD45.1 (JAX stock 002014- B6.SJL-Ptprca Pepcb/BoyJ) - strain most commonly used for the transplant of bone marrow cells.
CD45
NRG
BW difference
 Objective 1: To determine whether the use of DietGel76A™ will have a significant impact on clinical signs and the general survival rate of the
animals.
 Objective 2: To determine the potential differences between the used strains of mice in terms of sensibility/sensitivity and response to
gamma-irradiation.
 Objective 3: To investigate the differences in body weight (BW) between non-irradiated and sub-lethal irradiated cohorts, with the purpose to
assess whether a progressive weight loss is occurring, and to calculate the impact of this parameter over the animal wellbeing.
3
2
22.50
Objectives:
2
1
R² = 0.7565
The protocol for this experiment was reviewed and assessed by the Animal Care Committee (ACC) of the IRCM. All the animals used in this
experiment were cared for in compliance with the principles outlined in the current Guide to the Care and Use of Experimental Animals as published
by the Canadian Council on Animal Care.
1
17.50
c)
CD45
22.50
27.50
Animal Care Procedures
Group
Numbers
NRG
0.5
0
MATERIALS AND METHODS
C57
Irradiation +DietGel
1
10
a)
CD45
Irradiation w/t DietGel 22.50
Survival
30
DietGel 76A
Sub-lethal
Irrad
Group ‘’SD’’
Sub-lethal
Irrad only
Group ‘’S’’
Control
(no irrad)
Total Number
of
Animals
6
6
6
18
6
6
6
18
6
6
6
18
18
18
18
54
a) survival percentage, overall b) BW gain of the three strains, irradiated vs non-irradiated groups c) overall BW progression d) CD45.1 mice BW progression, e) NRG mice BW
progression, f) C57BL6 mice BW progression, g) NRG mice BW paradox progression, h) Variation of BW average after 40 days- on 3 strains, i) C57BL6 mice BW chart- irradiated vs non –
irradiated group.
DISCUSSION AND CONCLUSION
The analysis of the BW average gain between groups of irradiated animals treated and non-treated with DietGel showed a higher
variation (higher susceptibility) of NRG mice where the differences between two groups were most important. This is probably due to the
increased cellular sensitivity to ionizing radiation of the NRG mice , according to the JAX description and phenotype database entry
information for this strain.
 Objective 1: The impact over the survival rate of the animals was clearly demonstrated. Although the animals were sub-lethally
irradiated and no BMT was performed, the survival rate was exceptionally high.
 Objective 2: C57BL/6 irradiated mice responded the most intensely to the treatment with DietGel76A™, although they seemed to
be more resistant to the irradiation compared to the other two strains. In the case of NRG mice, which proved to be the most
sensitive to the irradiation procedure, the difference was less significant. However, it seemed that the withdrawal of DietGel76A™
after 21 days had a negative impact over the general health of the irradiated NRG mice.
 Objective 3: Following the irradiation of the three strains used in this experiment, we discovered that the most sensitive ones are
the NRG mice, followed by the C45.1 and the C57BL/6 mice.
Conclusion
The general objective of the experiment was to determine whether standard handling, sustained critical care and appropriate
Females(N)
medical treatment along with a dietary supplement could improve the survival rate of irradiated animals form three different
strains of mice.
Our hypothesis was that using DietGel76A™along with antibiotic treatment, strict handling and manipulation procedures, the
general mortality rate, as well as the onset of clinical signs between the treated animals and the control animals will be
significantly lower.
This hypothesis was confirmed for the C57BL/6 strain. The treatment with DietGel76A™ had a net positive impact on the
recovery of more irradiation sensitive strains (CD45.1 and mostly NRG). However, in our opinion, mice belonging to more
sensitive strains should receive DietGel76A™ for a longer period of time (42 days post irradiation).
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http://jaxmice.jax.org/strain/002014.html , http://jaxmice.jax.org/strain/007799.html
http://www.informatics.jax.org/greenbook/chapters/chapter22.shtml