Chesler1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedures
for
testing
nociception
and
pain
sensitivity
Data
References
Workflow
and
sampling
Workflow
Step |
Procedure
performed |
|
|
Equipment |
Age
(wk) |
Data
collected |
1 |
Each
mouse
is
tested
systematically
&
randomly
with
generated
unique
Latin-square
test
order;
body
weight
is
recorded
before
battery
testing |
- |
- |
Balance
scale |
7-15 |
|
2 |
Mice
pain
sensitivity
to
projected
high-intensity
heat
is
examined |
30
s |
3
|
High-intensity
heat
projector,
analgesia
meter,
timer |
7-15 |
latency
of
hindfeet
withdrawal |
3 |
Mice
pain
sensitivity
to
thermostatically
controlled
hot
plate
is
evaluated |
|
2 |
Thermostatically
controlled
metal
plate,
analgesia
meter,
timer |
7-15 |
latency
of
nociceptive
response |
4 |
Mice
pain
sensitivity
to
thermostatically
controlled
water
bath
is
investigated |
|
3
|
Thermostatically
controlled
water-bath,
analgesia
meter,
timer |
7-15 |
latency
of
tail
withdrawal |
5 |
Mice
pain
sensitivity
to
mechanically
applied
focal
stimulation
is
tested |
|
9 |
Calibrated
Von
Frey
nylon
monofilament
fibers |
7-15 |
latency
of
hindfeet
withdrawal |
6 |
Mice
pain
sensitivity
to
mechanically
applied
pressure
stimulation
is
examined |
|
1 |
Alligator
clips
with
rubber
cuffs |
7-15 |
latency
of
nociceptive
response |
Equipment
- Balance
scale
-
Stopwatch
- A
focused,
high-intensity
projector
lamp
beam
(IITC
Model
336
Plantar
Test
and
Tail-flick
Analgesia
Meter,
Woodland
Hills,
CA,
USA)
- 3/16th-inch
thick
glass
floor
within
small
(9
cm
L
×
5
cm
W
×
5
cm
H)
Plexiglas
cubicles
- Metal
surface
(IITC
Inc.,
Hotplate
Analgesia
Meter,
Woodland
Hills,
CA,
USA)
-
Transparent
Plexiglas
cylinder
(15
cm
D;
22.5
cm
H)
with
Plexiglas
lid
- Thermostatically
controlled
water
bath
(TW47)
by
Boekel
Scientific/Grant
Optima
Immersion
Circulator
Model
GR150
(Boekel
Scientific,
Feasterville,
PA,
USA)
-
Plexiglas-bound
arena
measuring
13.5
in
L
×
16
in
W
×
15
in
H
-
Alligator
clips
with
a
rubber
cuffs
around
each
jaw
(exerting
≈600
g
of
force)
-
Von
Frey
type
nylon
monofilament:
A
set
of
eight
calibrated
Von
Frey
fibers
(Stoelting,
IL,
USA),
ranging
from
0.067
to
9.33
g
of
force
Reagents,
supplies,
solutions
- Surface
cleanser
MB-10
(QuipLabs,
Wilmington,
DE,
USA)
- Paper
towels
Acclimation
to
test
conditions
Mice
are
allowed
to
acclimate
for
1
wk
to
new
housing
conditions.
Procedures
for
testing
nociception
and
pain
sensitivity
Hargreaves
paw
withdrawal
test
a.
Mice
are
allowed
to
habituate
for
a
period
of
2
hrs
on
a
3/16th-inch
thick
glass
floor
within
small
(9
cm
L
×
5
cm
W×
5
cm
H)
Plexiglas
cubicles.
b.
Following
a
period
of
habituation,
a
focused,
high-intensity
projector
lamp
beam
is
shone
from
below
onto
the
mid-plantar
surface
of
the
hindpaw
(Hargreaves
et
al,
1988).
c.
The
projector
lamp
is
set
to
10%
idle
intensity
(II10),
while
the
light-beam
is
aligned
to
the
mid-plantar
surface
of
the
left
paw.
d.
The
lamp
is
then
switched
to
25%
active
intensity
(AI25);
the
latency
to
respond
by
withdrawing
the
paw
away
from
the
light,
or
by
licking
of
the
paw,
is
recorded
with
the
internal
timer.
e.
The
lamp
is
turned
to
idle
intensity
after
30
s
and
the
paw
is
removed
from
from
the
platform
to
avoid
tissue
damage.
f.
Following
an
intra-trial
period
of
at
least
5
min,
the
above
process
is
repeated
for
the
right
hindpaw.
g.
Mice
are
tested
for
3
-
6
trials
depending
on
the
variance
observed
on
each
paw.
Only
the
three
tightest
latencies
are
averaged
and
recorded.
Hot
plate
test
a.
Mice
are
allowed
to
habituate
to
the
testing
room
for
30
min.
b.
Following
a
period
of
habituation,
the
mice
are
placed
on
the
center
of
a
hot
metal
surface
maintained
at
54
±
0.2°C
temperature
(HP52)
within
a
transparent
Plexiglas
cylinder
(15
cm
D;
22.5
cm
H)
equipped
with
a
Plexiglas
lid
and
a
built-in
timer
that
is
started
immediately.
c.
The
latency
to
respond
with
a
jump,
or
hindpaw
lick
or
shake/flutter
is
measured
to
the
nearest
0.1
s.
d.
Two
trial-latencies
are
recorded
per
mouse
with
a
30
s
intra-trial
interval
and
a
maximum
trial
duration
of
30
s.
e.
To
prevent
tissue
injury
from
the
hot
plate,
each
mouse
is
promptly
removed
after
30
s.
f.
The
apparatus
is
thoroughly
cleansed
with
MB-10
between
each
testing.
Notes
from
submitting
PI:
"This
test
occurs
approximately
2–3
h
after
the
completion
of
the
open
field
test.
The
lights
in
the
testing
area
are
turned
off
at
least
an
hour
prior
to
testing
and
animals
are
allowed
to
sit
undisturbed
in
the
darkened
room.
A
lamp
(15
W
bulb)
behind
the
hot
plate
(Hotplate
Analgesia
Meter,
Model
39,
IITC,
Inc.)
is
faced
away
from
the
hot
plate
surface.
A
mirror
is
placed
behind
the
hot
plate
so
that
the
experimenter
can
observe
the
animal."
Tail
withdrawal
test
a.
Mice
are
allowed
to
habituate
for
30
min
to
the
testing
environment.
b.
Each
mouse
is
lightly
restrained
in
a
denim
pocket
while
the
distal
half
of
it's
tail
is
dipped
into
a
thermostatically
controlled
recirculating
waterbath
at
47.0
±
0.1°C)
(TW47)
temperature.
c.
Latency
to
respond
to
the
heat
stimulus
by
vigorous
flexion
of
the
tail
is
measured
with
a
stopwatch.
d.
Each
mouse
is
given
3-5
trials
with
an
inter-trial
interval
of
10
s,
and
a
maximum
trial
duration
of
30
s.
e.
To
prevent
tissue
injury
each
mouse's
tail
is
promptly
removed
from
the
hot
water
after
30
s.
f.
Latency
performance
of
the
last
3
trials
are
recorded.
Tail
clip
test
a.
Mice
are
allowed
to
habituate
for
30
min
to
the
testing
environment.
The
enclosure
is
a
Plexiglas-bound
arena
measuring
13.5
in
L
×
16
in
W
×
15
in
H.
b.
The
front
of
the
arena
is
opened
and
aligned
with
the
leading
edge
of
the
table,
such
that
the
experimenter
can
easily
restrain
and
release
mice
quickly.
c.
All
mice
are
lightly
restrained
in
a
denim
pocket,
while
an
a
rubber
cuff
protected
alligator
clip
(exerting
≈600
g
of
force)
is
applied
to
the
tail
1
cm
from
the
base
and
vertically
oriented
with
respect
to
the
table.
d.
The
mouse
is
immediately
removed
from
the
holder,
and
the
latency
to
lick,
bite,
or
grab
the
clip
or
bring
its
head
within
1
cm
of
the
clip
is
measured
with
a
stopwatch
to
the
nearest
0.1
s,
after
which
the
clip
is
immediately
removed.
e.
Each
mouse
is
tested
only
once
with
maximum
trial
duration
of
60
s.
f.
To
prevent
tissue
injury
the
tail
clip
is
promptly
removed
after
60
s.
g.
The
enclosure
and
clip
are
thoroughly
cleansed
with
MB-10
between
each
mouse.
Von
Frey
test
a.
Mice
are
allowed
120
min
of
habituation
to
the
same
Plexiglas
enclosure
(9
cm
L
×
5
cm
W
×
5
cm
H)
on
a
wire-mesh
floor
(aquarium/vivarium
top)
instead
of
a
glass
floor.
b.
For
assessment
of
mechanical
sensitivity
thresholds,
mice
are
tested
with
von
Frey
type
nylon
monofilaments.
c.
A
set
of
8
calibrated
Von
Frey
fibers,
ranging
from
0.067
to
9.33
g
of
force,
are
applied
to
the
plantar
surface
of
the
hindpaw
until
they
bend.
•
1
of
a
series
of
8
Von
Frey
fibers
with
logarithmically
increasing
stiffness
(0.067
to
9.33
g)
is
used
to
stimulate
the
hindpaw.
•
The
Von
Frey
fiber
is
positioned
perpendicular
to
the
plantar
surface,
enough
pressure
is
applied
to
cause
slight
bending
against
the
paw,
and
held
in
place
for
approximately
6-8
s.
•
Only
when
behavioral
responses
to
previous
stimuli
have
resolved
that
the
series
of
stimuli
are
presented
at
several
seconds
intervals.
•
If
the
paw
is
sharply
withdrawn,
a
positive
response
is
noted.
•
If
the
mouse
flinch
immediately
when
the
fiber
is
removed,
a
positive
response
is
noted
.
•
If
a
mouse
walks
away,
the
response
is
considered
ambiguous,
and
the
stimulus
is
repeated.
d.
The
threshold
force
required
to
elicit
withdrawal
of
the
paw
(median
50%
paw
withdrawal)
is
determined
using
the
up–down
method
(Chaplan
et
al.
1994).
•
Testing
is
started
with
the
2.0
g
fiber,
in
the
middle
of
the
series.
•
Stimuli
are
always
presented
in
a
consecutively,
either
ascending
or
descending.
•
If
there
is
no
hindpaw
withdrawal
response
to
the
first
selected
fiber,
a
stronger
stimulus
is
tried;
if
a
hindpaw
withdrawal
is
elicited,
the
next
weaker
stimulus
is
selected.
•
Optimal
threshold
calculation
by
this
up-down
method
requires
6
withdrawal
responses
in
the
immediate
vicinity
of
the
50%
threshold.
•
Series
of
similar
responses
may
be
generated
as
the
threshold
is
approached
from
either
up
or
down
direction.
•
Although
all
responses
are
noted,
counting
of
the
critical
6
data
points
does
not
begin
until
the
response
threshold
is
first
crossed,
at
which
time
the
2
responses
straddling
the
threshold
are
retrospectively
designated
as
the
first
2
responses
of
the
series
of
6.
•
Four
additional
responses
to
the
continued
presentation
of
stimuli
that
were
varied
sequentially
up
or
down,
based
on
the
rat's
response,
constituted
the
remainder
of
the
series.
•
Thus,
the
number
of
actual
responses
collected
using
this
paradigm
can
vary
from
a
minimum
of
4
(in
the
case
of
paw
withdrawal
sequentially
to
the
4
hairs
in
the
descending
range
2.0-0.4
g:
threshold
lies
below
the
range
of
actual
stimuli)
to
a
maximum
of
9
(in
the
case
of
the
first
withdrawal
occurring
on
the
fifth
ascending
stimulus
presentation
at
15.1
g,
followed
by
elicitation
of
4
additional
responses,
assuming
that
withdrawals
continue
to
occur
at
or
below
15.1
g).
•
In
cases
where
continuous
positive
or
negative
responses
are
generated
to
the
exhaustion
of
the
stimulus
set,
values
of
15.00
g
and
0.25
g
are
assigned
respectively.
•
The
resulting
pattern
of
positive
and
negative
responses
is
tabulated
using
the
convention,
X=
withdrawal;
0
=
no
withdrawal,
and
the
50%
response
threshold
is
interpolated
using
the
following
formula:
50%
g
threshold
=
(10
[Xf+κδ)/10,000
•
Xf
=
value
(in
log
units)
of
the
final
Von
Frey
fiber
used
•
k
=
tabular
value
(see
Appendix)
for
the
pattern
of
positive/negative
responses
•
δ
=
mean
difference
(in
log
units)
between
stimuli
(here,
0.224)
e.
A
maximum
of
9
trials
are
required
for
each
paw
with
4
trials
performed
after
the
1st
response
cross-over.
f.
Trials
begin
with
the
left
hindpaw
and
then
to
the
right
hindpaw
after
all
mice
in
the
enclosure
receive
the
stimulus.
g.
An
intra-trial
period
of
150
s
is
used
between
left
and
right
paw.
|
|
Test age:
7-15wks
