Institution
Name
Address
066 – UiB – Vivarium
Vivarium UiB, Haukeland Sykehus, 5021 Bergen
Telephone
E-mail
Responsible person
Applicant
Application Date
Aurora Brønstad
Mohummad Aminur Rahman
18.09.2012
General Information
ID
XXXX
Applicant reference #
Working title
Description
Animal Species
Applicant’s institution
Application type
Epigenetic regulation in glioblastoma multiforme.
Aberrant epigenetic landscapes and their involvement in genesis
and progression of tumors, as well as in treatment responses and
prognosis, indicate one of the most emerging fields in cancer
research. Glioblastoma Multiforme (GBM) is lethal cancers
which infiltrate the surrounding brain structure extensively, with
short survival and new therapy is urgently needed. Studies show
that cancer associated fibroblasts (CAFs) actively foster tumor
growth, but the role of glial cells in brain tumor is unknown. In
this experiment we will implant human glioblastoma biopsies in
NOD/Scid mice expressing green fluorescent protein (GFP), and
establish glioma phenotypes representing stages before and after
the onset angiogenesis. Tumor associated glial cells (TAGs) will
be sorted by fluorescence activated cell shorting (FACS) and
DNA will be isolated to check the DNA methylation patterns.
Mammals - rodents (Rodentia) - Mouse (Mus musculus)
066 – UiB – Vivarium
New Application
Acute experiments
No
Previous experience with similar
experiments
No
Research is financed by
Other funding
Planned start
25.11.2012
Planned end
24.11.2013
Offentlighet
Contains the application data that should be
exempt from public disclosure?
Article
If yes, state what information these apply
and provide a rationale for why it should be
exempt from public disclosure. (cf. of § 5a
and Administration Act § 13 subsection 2)
No
Background and Purpose
Provide a brief description, in accessible
language ailment shape of the background
and purpose of the experiment and, specify
any hypothesis to be tested. Enter separately
if special laws / requirements from public
authorities require that the experiment
should be performed:
Cancer cells undergo massive alterations to their DNA
methylation pattern that result in aberrant gene expression and
malignant phenotypes. Previous studies in our laboratory
showed that mice co-implanted with TAGs and glioma cells
develop tumors faster than mice receiving only glioma cells or
glioma cells with unconditioned normal glial cells. Moreover,
whole genome microarray analysis of TAGs and normal glial
cells showed a gene expression profile distinct from normal glial
mice cells. It would be interesting to see the DNA methylation
patterns of tumor associated glial cells (TAGs) comparing with
the normal glial cells.
Calculation of the number of animals
Provide a rationale for the number of
animals. If there is uncertainty about
population size will be conducted pilot
experiments, jf § 13 It is recommended to
seek help from a statistician himself if you
do not feel competent. Provide an overview
of all experimental groups and group sizes.
Which method is used for calculating the
number of animals? Describe different
method and justify by not applicable
We will use 27 animals that will be divided into 3 groups, 3
parallels, 3 animals each groups.
Group 1: Control group, 9 animals.
Group 2: Tumor sample 1 (Patient 1), 9 animals.
Group 3: Tumor sample 2 (Patient 2), 9 animals.
We will need 3 parallels in each group in order to be able to
perform statistical analysis.
Alternatives / 3R
Replacement: Why cannot replace this
experiment with alternative methods
without the use of animals? What options or
considered and why they are rejected?
Which databases were searched and what
keywords were used?
Reduction: What steps have you taken to
reduce the number of animals in this
experiment?
It is not possible to recapitulate the interaction between
endothelial cells, tumor cells and other tumor-associated cells in
culture in a physiologically correct manner in a therapy trial.
Pubmed, science direct, google schoolar
GBM biopsy xenograft, DNA methylation.
The number of animals needed to detect a significant biological
effect will also reduce the variation in the results. It is therefore
important to standardize the different steps of the experimental
setup: 1) Tumor Burden: We are implanting same number of
tumor cells. 2) Procedure: We standardize implantation site in
the brain as much as possible. 3) Animals: Use as much as
possible animals with small differences in the age and weight 4)
Two operators do all implantations. 5) Standardize the
remaining experimental conditions- e.g. do all implantations in
the same group on the same day, and in the same session.
Refinement: Is it made any improvements in
the experimental setup or protocol, what
you will emphasize, that makes this a more
delicate test for the animals than what has
been normal for similar experiments?
(Keyword anesthesia/ pain management,
endpoints, environmental enrichment,
surgical technique, etc.):
1) We replace xylocain (1%) with Marcaine (0.5%) for longer
postoperative analgesia. 2) We routinely perform stereotactic
implantation to increase precision and gentleness of the
procedure.
3)
We
use
the
same
microscope
4) We prepare the procedure as a sterile procedure in the same
manner as the operating room with sterile cover, autoclaving of
all equipment and their own ventilation of the operating field
(extraction). 5) We have two people operating together to reduce
the operation time (One does implantation, while another makes
access and closure). 6) We have postoperative awakening in an
incubator set at 33-35 degrees C.
Method description
Preparation of the animals prior to solve the
experiment any innfangingsmetode, fixation
method, identification method, shipping
method, etc..:
Studies will be conducted on male and female homozygous
NOD/Scid mice bred and maintained in an isolation facility in a
pathogen free environment on a standard 12/12 h day and night
cycle. Animals are fed a standard sterilized pellet diet and
provided sterile tap water ad libitum. 5 tumour spheroids (250350 ìm in diameter) will be selected under a light microscope.
The animals will be anaesthetized with Isofluran gass and the
head secured in a stereotactic frame (Benchmark; Neurolab,St
Louis, MO). Marcaine injection will be done locally, and a short
longitudinal incision will be made in the scalp exposing the
calvarium.
Which intervention (surgery, administration
of the test substance, physical treatments
mm.) Shall be made on the animal during
the experiment (possibly with trains
drawing of surgical techniques,
experimental protocol to supplement mm):
A burr-hole will be made 0,5 mm posterior to the bregma and
1,5 mm to the right of the sagittal suture using a micro-drill. A
Hamilton syringe with inner diameter of 810 ìm will be
introduced to a depth of 1,5 mm below the brain surface, and the
spheroids will slowly be injected and the syringe left in place for
3 min before withdrawal. The skin will be closed with an
Ethilon 3- 0 suture. The tumours are allowed to grow for 4-6
weeks. Whereupon they will be subjected to collect the tumor,
the tumor bearing brains will then be removed for further
analysis. Animals will be sacrificed by CO2 inhalation.
What parameters are painted during the
trial?
Briefly describe the experimental groups
and any sentinel (Please enclose a table):
Tumor growth will be monitored by MR.
We will use 27 animals that will be divided into 3 groups, 3
parallels, 3 animals each groups.
Group 1: Control group, 9 animals.
Group 2: Tumor sample 1 (Patient 1), 9 animals.
Group 3: Tumor sample 2 (Patient 2), 9 animals.
We will need 3 parallels in each group in order to be able to
perform statistical analysis.
Set the monitoring and surveillance of the
animals during surgery, rats operation and
during the rest of the test period otherwise:
Set methods of euthanasia and why this
method is selected, using compositions
provide generic name and trade name and
dosage:
Criteria for humane endpoints:
In our experience, there is no mortality associated with the
stereotactic implantation of the tumors in the brain. Whereupon
they will be subjected to collect the tumor, the tumor bearing
brains will then be removed for further analysis. Animals will be
sacrificed by CO2 inhalation.
CO2 inhalation
Weakness, weight loss and neurological symptoms.
Action:
Euthanasia
Species
Mammals - rodents (Rodentia) - Mouse (Mus musculus)
Animals (Art, sedation and pain)
Line/Tribe
Sex
Number
Weight
Age
Number of animals for reuse (§ 15)
Experience with this species
Duration of each animal (d, h, min)
NOD/Scid
Both
45
20-45 g
From 6 weeks
Not possible
Yes
90, 0, 0
Animals with an aberrant phenotype
Should work with GM animals?
Yes
Notify the Social and Health Services
No
Reported from the Directorate for Nature
No
If GM, will the animals bred at the
institution?
Have animals inherited disease / disorder
that can affect their welfare (examples:
diabetes, autoimmune disease, an increased
incidence of tumors, disorders of the
musculoskeletal system, dental defects
etc.)?
What measures / behandlning be taken to
ensure the welfare of animals with
hereditary / congenital disease / disorder
mentioned above, and when do you expect
that it will be necessary?
Yes
The animals have reduced B- and T-cell numbers, and a reduced
immune system. Despite this they can tolerate tumor
implantation well, and do rarely get post-operative infections.
Such measures will not be needed
Sedation, anesthesia, analgesia
Period
Type
Prescribing
Induction Dose
(mg/kg)
Maintenance Dose
(mg/kg)
Administration-way
Under
Anesthesia
Isofluran
inhalation by mask
Under
Anesthesia
Marcain
Subcutaneous
After
Anesthesia
Marcain
Other medications (all other drugs / test
substances used)
Neuromuscular blockers to be used
Anesthesia dosage is initially 5% isoflurane, 150ml air / min at
induction and then lowered to 1% isoflurane in 150 ml air / min
for maintenance. We use 2.5 mg/ml Marcaine.
No
Justification for the use of neuromuscular
blocking agent:
Pain and discomfort
Analgesia or not applicable
Reason for analgesia omitted experiment
considered a mean significant / persistent
pain or discomfort.
Justification of ratings
No
We have a long experience in establishing human brain tumors
in immune-deficient mice. The brain represents an organ that
does not have pain receptors. There is therefore no need for
analgesia.
No
Intensity of pain
Little
Duration of pain
2 hours
Justification for the choice of animal model
Provide a rationale for the choice of animal
model, see Regulations § 8 - animal, line,
sex, age, special features, Gene
modifications
We have to use an animal model that will accept
xenotransplantation of human tumors. 15 years of experience
has shown us that the mice model is one of the best and most
reliable models to establish brain tumor xenografts.