Nelson1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
and
supplies
Reagents
and
solutions
Procedures
Data
References
Workflow
and
sampling
Workflow
|
Procedure
performed |
|
|
|
|
Mice
housed
5
per
cage,
according
to
sex
and
strain
|
|
|
- |
|
Mice
divided
into
2
groups.
Cohort
1:
ad
libitum
(AL)
diet
and
housed
5
per
cage;
cohort
2:
40%
dietary
restriction
(DR;
60%
of
strain-specific
AL
intake)
and
housed
6
per
cage
|
|
|
- |
|
At
12
mo
of
age,
rations
fixed
to
avoid
tracking
the
reduction
of
food
intake
that
can
occur
during
aging |
|
|
- |
4 |
Mice
weighed |
AL,
DR |
15-17 |
body
weight |
5 |
Whole-body-composition
determined
(no
sedation) |
AL,
DR |
15-17 |
lean
and
fat
tissue
mass |
|
Mice
aged
for
maximum
lifespan
determination
(sick
or
moribund
mice
sacrificed)
|
|
|
|
Equipment
and
supplies
- Balance
scale
- ID
implantable
chips:
AVID
Microchip
ID
Systems
(Folsom,
LA,
USA;
catalog
AVID3002)
- Transponders
(rice-grain-size,
~0.1
g)
(Bio
Medic
Data
Systems,
Seaford,
DE)
for
mouse
identification
- QMR
machine
utilizing
NMR
(EchoMRI;
Echo
Medical
System,
Houston
TX
USA)
- Plastic
restrainer
tubes
for
QMR
machine
Reagents
and
solutions
- Superglue
(Loctite
gel,
purchased
locally,
or
Nexaband
S/C,
purchased
from
Abbott
Laboratories,
North
Chicago,
IL,
USA).
- Harlan-Teklad
7912
diet
(>19%
crude
protein,
>5%
crude
fat,
<5%
crude
fiber);
nutrient
requirements
for
laboratory
mice
and
nutrient
composition
of
Harlan-Teklad
7912:
| Diet
Composition |
Mouse
Requirementa |
Harlan-Teklad
7912 |
|
|
Calcium
(%) |
0.5 |
0.98 |
|
Phosphorus
(%) |
0.3 |
0.66 |
|
Sodium
(%) |
0.05 |
0.32 |
| Chlorine
(%) |
0.015 |
0.54 |
| Potassium
(%) |
0.2 |
0.83 |
| Magnesium
(%) |
0.05 |
0.25 |
|
Iron
(mg/kg) |
35 |
284 |
| Manganese
(mg/kg) |
10 |
93 |
| Zinc
(mg/kg) |
10 |
64 |
| Copper
(mg/kg) |
6
|
23 |
| Iodine
(mg/kg) |
0.15 |
2.61 |
| Cobalt
(mg/kg) |
ND |
0.72 |
| Selenium
(mg/kg) |
0.15 |
0.16 |
|
| Vitamin
A
(IU/g) |
2.4 |
29.6 |
| Vitamin
D3
(IU/g) |
1.0 |
2.4 |
| Vitamin
E
(IU/kg) |
32 |
114 |
| Choline
(mg/kg) |
2.0 |
2.2 |
| Niacin/Vitamin
B3
(mg/kg) |
15 |
100 |
| Pantothenic
Acid/Vitamin
B5
(mg/kg) |
16 |
87 |
| Pyridoxine/Vitamin
B6
(mg/kg) |
8 |
17 |
| Thiamine/Vitamin
B1
(mg/kg) |
5 |
95 |
| Menadione
(mg/kg) |
ND |
8.4 |
| Folic
Acid/Vitamin
B9
(mg/kg) |
0.5 |
6.7 |
| Biotin
(mg/kg) |
0.2 |
0.8 |
| Vitamin
B12
(mcg/kg) |
10 |
91 |
a
National
Research
Council
requirements
for
mice
(Nutrient
Requirements
for
Laboratory
Animals,
1995).
ND
=
not
determined.
Procedures
I.
Mouse
identification
a.
For
accurate
and
reliable
identification,
transponders
are
used
(see
above
Figure).
b.
Each
mouse
is
briefly
anesthetized
for
1–2
min
with
isoflurane
inhalation
administered
either
via
nose
cone
or
with
an
instrument
designed
for
small
animal
anesthesia.
c.
An
electronic
ID
chip
is
then
implanted
using
a
sterile
syringe
beneath
the
dorsal
skin
between
the
shoulder
blades.
d.
A
portion
of
the
distal
tail
(1
cm)
is
sampled
and
frozen
for
later
analysis
of
DNA
polymorphisms.
e.
The
injection
wound
is
closed
with
a
drop
of
superglue
before
each
mouse
is
allowed
to
recover
from
the
anesthesia.
f.
Mice
without
transponders
are
given
a
left-ear
or
right-ear
punch
for
identification.
Submitting
PI
notes:
"An
ANOVA
of
lifespan
using
diet,
strain,
and
transponder
as
between
group
factors
indicated
no
main
effect
of
the
transponders
(p=0.22)
and
no
interaction
with
diet
or
strain
or
both
(p>0.8)."
II.
Longevity
feeding
a.
At
2–5
mo
of
age
mice
are
fed
ad
libitum
(AL)
or
40%
diet
restriction
(DR;
60%
of
strain-specific
AL
intake).
b.
DR
rations
are
calculated
on
the
basis
of
AL
food
intake
measured
weekly
for
each
strain
and
adjusted
for
wastage
(Ikeno
et
al.,
2005).
To
measure
food
consumption,
the
amount
of
chow
removed
from
the
cage
hopper
and
the
spillage
(the
chow
on
the
bottom
of
the
cage)
are
weighed
weekly.
Actual
food
consumed
is
calculated
by
subtracting
the
spillage
from
the
chow
removed
from
the
hopper.
c.
DR
mice
are
fed
approximately
1h
before
the
start
of
the
dark
phase
of
a
12-hour
light/dark
cycle
(lights
on
at
0050h).
d.
Rations
are
weighed
to
the
nearest
0.1
g
and
delivered
manually
to
the
bottom
of
the
cage,
rather
than
the
food
hopper,
to
make
gnawed
pellets
easier
to
grasp
and
more
accessible.
e.
The
AL
and
DR
cages
are
maintained
side
by
side
in
alternating
columns
of
AL
and
DR
to
minimize
the
potential
for
shelf-level
variation
in
AL
food
intake.
f.
Because
AL
mice
exhibit
a
substantial
decline
in
their
food
intake
with
age,
the
DR
rations
are
kept
constant
and
fixed
by
12
mo
of
age.
III.
Body
weight
and
body
composition
a.
At
15-17
mo
of
age,
mice
are
immobilized
using
a
plastic
restrainer
tubes
(no
sedation)
placed
in
the
QMR
machine.
b.
Whole-body-composition
analysis
is
conducted,
and
AL
and
DR
mice
are
analyzed
over
the
same
time
period.
c.
From
scanned
images,
total
body
fat
mass
and
lean
mass
are
calculated.
d.
Body
weight
is
recorded
using
a
balance
scale.
IV.
Longevity
mouse
monitoring
a.
When
half
of
the
mice
from
the
same
diet
and
strain
die,
the
remaining
mice
are
consolidated
in
a
single
cage
in
keeping
with
the
original
cage
density
(5
for
AL,
6
for
DR).
b.
Mice
are
examined
at
least
daily
for
signs
of
ill
health
and
any
mouse
found
dead
is
noted.
c.
Moribund
mice
are
euthanized
(a
mouse
is
considered
severely
moribund
if
it
exhibits
more
than
one
of
the
following
clinical
signs:
(i)
inability
to
eat
or
drink;
(ii)
severe
lethargy,
as
indicated
by
reluctance
to
move
when
gently
prodded
with
forceps;
(iii)
severe
balance
or
gait
disturbance;
(iv)
rapid
weight
loss
over
a
period
of
1
wk
or
more;
or
(v)
an
ulcerated
or
bleeding
tumor).
d.
The
age
is
recorded
as
the
best
estimate
of
its
lifespan.
Definitions
and
calculations
AL:
ad
libitum
40%
dietary
restriction
(DR):
60%
of
the
volume
consumed
by
ad
libitum
(AL)
fed
mice
(determined
for
each
strain)
NMR:
nuclear
magnetic
resonance
used
in
whole-body-composition
analysis
QMR:
Quantitative
magnetic
resonance
(QMR)
system
measures
whole
body
fat
mass,
lean
tissue
mass,
free
water,
and
total
body
water
in
live
mice,
without
the
need
for
anesthesia
or
sedation,
in
less
than
1
min.
Data
collected
by
investigator
- body
weight
- lean
tissue
mass
- fat
tissue
mass
- lifespan:
age
in
days
MPD
computed
percent
fat
|
|
Test age:
max lifespan
