1. Age-­‐related
changes
in
the
primary
afferent
function
in
vitro
Liang
Huang,
Ratan
Banik
New
Jersey
Neuroscience
Institute
Abstract
The
altered
pain
perception
and
the
cutaneous
nociception
elicited
by
noxious
stimuli
to
the
skin
during
senescence
are
not
well
understood,
and
it
is
thought
that
this
could
in
part
be
due
to
changes
in
peripheral
pain
sensing
processes.
We
systematically
examined
cutaneous
nociceptor
responses
and
nociceptive
behaviors
in
young
(2-­‐6
months)
and
in
aged
(18-­‐26
months)
F334/N
rats.
C-­‐fiber
nociceptors
in
the
skin
were
identified
by
mechanical
stimulation,
and
extracellularly
recorded
from
hind
paw
skin-­‐saphenous
nerve
preparations
in
vitro.
The
aim
of
the
present
study
was
to
investigate
the
activities
of
aged
skin
nociceptors
systematically
to
mechanical,
chemical
stimuli,
and
to
compare
with
the
data
from
young
animals.
Mechanical
threshold
measured
by
a
ramp
mechanical
stimulus
in
the
aged
skin
was
significantly
higher
than
that
in
the
younger
skin.
The
latency
to
chemical
stimulations
tended
to
be
longer.
In
addition,
the
magnitude
of
the
chemical
response
during
the
60s
chemical
stimulus
was
not
significantly
different.
In
contrast,
the
numbers
of
total
net
discharges
induced
by
chemical
(bradykinin,
prostaglandin,
serotonin,
histamine)
stimuli
were
not
different
with
the
different
ages.
After
sensitization
by
chemicals,
the
young
rats
displayed
a
stronger
and
longer
mechanosensitization.
This
showed
for
the
first
time
that
not
only
receptive
properties
of
afferent
terminals
but
also
mechanical
sensitizations
by
chemicals
in
axons
are
changed
in
aged
rats.
These
results
showed
decreased
mechanical
and
chemical
responses
in
skin
C-­‐afferents
in
the
aged
rats.
Introduction
With
advancing
age,
a
decline
in
the
sensation
is
well
reported
to
occur.
Ageing
influences
on
morphological
and
functional
features
of
cutaneous
mechanical
transducers
and
mechanosensitive
ion
channels,
sensory
innervation,
neurotransmitters
and
even
vascular
system
required
to
ensure
efferent
function
of
the
afferent
nerve
fibres
in
the
skin.
This,
in
conjunction
with
effect
of
ageing
on
the
skin
per
se
and
central
nervous
system,
could
significantly
affect
the
skin
sensation
among
the
ageing
population.
However,
little
is
known
about
the
peripheral
neural
mechanisms
of
skin
nociception
in
the
aged.
Ageing
is
associated
with
reductions
of
the
principal
functions
of
the
skin,
including
protection,
excretion,
secretion,
absorption,
thermoregulation,
pigmentogenesis,
and
regulation
of
immunological
processes
and
wound
repair.
Ageing
is
also
associated
with
a
progressive
decline
in
cutaneous
thermal,
vibratory
and
mechanical
sensory
perception
(Guergova
S.,
Thermal
sensitivity
in
the
elderly:
a
review
Ageing
Res.
Rev.,
10
(2011),
Lin
Y.H.,
Influence
of
aging
on
thermal
and
vibratory
thresholds
of
quantitative
sensory
testing.
J.
Peripher.
Nerv.
Syst.,
2005
and
Taguchi,
2010).
However,
the
change
with
age
in
pain
perception
in
humans
and
the
nociceptive
behaviors
in
animals
elicited
by
noxious
stimuli
to
the
skin
are
not
well
understood,
and
little
is
known
about
the
peripheral
neural
mechanisms
of
cutaneous
nociception
in
the
aged
and
responses
to
mechanical
stimulation
and
to
inflammatory
soup

2. were
not
recorded.
The
sensitizations
of
mechanical
response
by
inflammatory
soup
from
different
age
groups
remain
unclear.
To
date,
nearly
all
attempts
to
characterize
aged
afferent
fibers
have
utilized
structural,
biochemical,
or
molecular
measures1-­‐12
.
Morphologic
studies
reported
several
abnormalities
after
aging
such
as
demyelination,
axonal
atrophy,
reduction
in
the
expression
of
cytoskeletal
proteins5,9,10
.
Biochemical
studies
found
reduction
of
neuropeptide
expression13
and
molecular
studies
found
reduction
in
the
expression
of
the
molecules
necessary
for
transduction
of
natural
stimuli6,7,11
.
Using
an
in
vitro
skin-­‐saphenous
nerve
preparation,
single-­‐fiber
recordings
were
made
from
mechano-­‐
heat
sensitive
C-­‐fiber
nociceptors
innervating
rat
glabrous
hind
paw
skin,
and
their
responses
were
compared
with
those
obtained
from
different
age
groups.
Responses
to
mechanical
stimulation
and
to
inflammatory
soup
were
tested.
The
sensitizations
of
mechanical
response
by
inflammatory
soup
from
different
age
groups
were
also
investigated.
Methods
Animals
Experiments
were
performed
on
52
male
F344/N
rats
of
various
ages.
Two
months
(n=13),
6
months
(n=22),
18
months
(n=6),
and
26
month
(n=11)
old
rats
were
purchased
from
National
Institute
of
Aging,
Bethesda,
Maryland,
USA.
Two
to
four
animals
were
placed
in
plastic
cages
with
sawdust
bedding
and
housed
in
a
climate-­‐controlled
room
under
a
14/10
hr
light/dark
cycle.
The
Animal
Care
and
Use
Committee
at
The
Seton
Hall
University,
South
Orange,
New
Jersey,
USA
has
approved
experiments,
and
the
animals
were
treated
in
accordance
with
the
Ethical
Guidelines
for
Investigations
of
Experimental
Pain
in
Conscious
Animals.
Organ
bath
Electrophysiological
recordings
were
performed
in
animals.
Animals
were
killed
using
CO2
inhalation;
then
hairy
skin
of
the
rat
hind
paw
and
its
intact
saphenous
nerve
were
dissected
free
from
muscles
and
tendons.
The
preparation
was
then
placed
in
an
organ
bath
and
was
continuously
super
fused
with
a
modified
Krebs-­‐Henseleit
solution
(in
mM:
110.9
NaCl,
4.8
KCl,
2.5
CaCl2,
1.2
MgSO4,
1.2
KH2So4,
24.4
NaHCO3,
and
20
glucose),
which
was
saturated
with
a
gas
mixture
of
95%
O2
and
5%
CO2.
The
temperature
of
the
bath
solution
was
maintained
at
34
±
1°C.
After
dissection,
the
preparation
was
placed
with
‘epidermal
side
down’.
The
nerves
attached
to
the
skin
were
drawn
through
one
small
hole
to
the
second
chamber,
which
was
filled
with
liquid
paraffin.
The
nerves
were
placed
on
a
fixed
mirror,
their
sheaths
removed
and
nerve
filaments
repeatedly
teased
to
allow
single
fiber
recording
to
be
made
by
using
double-­‐platinum
electrodes
(one
for
recording
and
another
for
reference).
Single
nociceptive
afferent
fibers
were
recorded
extracellularly
with
a
differential
amplifier
(DAM50,
Harvard
Apparatus,
Holliston,
MA).
Neural
activity
was
amplified
and
filtered
using
standard
techniques.
Amplified
signals
were
led
to
a
digital
oscilloscope
and
an
audio
monitor
and
fed
into
PC
computer
via
a
data
acquisition
system
(spike2/CED1401
program).
Action
potentials
collected
on
a
computer
were
analyzed
off-­‐line
with
a
template
matching
function
of
spike
2
software.
Identification
of
afferents

3. The
search
strategy
was
mechanical
stimulation
by
a
fire-­‐polished
glass
rod;
thus,
mechanosensitive
afferents
were
characterized.
Only
units
with
a
clearly
distinguished
signal
to
noise
ratio
were
further
studied.
Rapidly
adapting,
low
threshold
A-­‐β
and
D-­‐hair
fibers
were
not
studied.
After
the
initial
assessment,
fibers
were
evaluated
for
their
responsiveness
to
controlled
mechanical
stimuli
and
a
cocktail
of
chemicals,
previously
termed
as
‘inflammatory
soup’.
In
this
study,
ingredients
of
this
soup
and
concentrations
were
different
from
other
studies
and
the
pH
was
normal
(7.4).
Aliquots
(20
µl)
of
chemical
cocktail
were
prepared
by
combining
bradykinin,
serotonin,
and
histamine
dissolved
in
distilled
water
with
prostaglandin
E2
dissolved
in
dimethyl
sulfoxide
(DMSO)
and
stored
at
-­‐20°C.
The
aliquots
were
diluted
to
final
concentration
(10
µM)
in
neutral
(7.4)
Krebs’
solution
on
the
day
of
the
experiment.
All
chemicals
were
obtained
from
Sigma-­‐Aldrich
(St.
Louis,
MO).
The
decision
to
use
10
µM
concentration
of
the
soup
was
based
on
the
results
of
published
studies
(14,15
).
Conduction
Velocity
and
Fiber
Categorization
In this study we concentrated on the C-fiber nociceptors. The
conduction
velocity
was
always
measured
at
the
end
of
the
experiment
to
avoid
damage
to
the
receptive
field
or
alteration
of
fiber
properties.
The
conduction
velocity
of
the
axon
was
determined
by
monopolar
electrical
stimulation
through
an
epoxy-­‐coated
electrode.
The
electrical
stimulation
(1-­‐20
V
at
0.2-­‐1
Hz
for
0.5-­‐2
ms)
was
delivered
at
the
sensitive
spot
of
a
receptive
field.
The
intensity
of
the
stimulus
started
form
0.1
V
and
gradually
increased
until
the
similar
shape
spike
appeared.
The
distance
between
receptive
field
and
the
recording
electrode
(conduction
distance)
was
divided
by
the
latency
of
the
action
potential
(stimulus
artifact
to
the
appearance
to
spike).
The
fibers
were
classified
using
criteria
from
Leem
et
al.16
Afferent
fibers
conducting
slower
than
2.5
m/s
were
classified
as
C-­‐fibers,
those
conducting
between
2.5
m/s
and
24
m/s
as
Aδ-­‐fibers,
and
those
conducting
faster
than
24
m/s
as
Aβ-­‐fibers.
Units
were
classified
as
mechanosensitive
nociceptors
on
the
basis
of
their
graded
response
throughout
the
innocuous
and
noxious
range
of
mechanical
force
stimuli.
Rapidly
adapting
fibers
were
not
studied.
Feedback
–controlled
mechanical
stimulation
To
measure
quantitative
mechanosensitivity,
a
servo
force-­‐controlled
mechanical
stimulator(Series
300B
Dual
Mode
Servo
System;
Aurora
Scientific,
Aurora,
Ontario,
Canada)17
were
used.
A
flat
and
cylindrical
metal
probe
(tip
diameter,
0.7
mm)
attached
to
the
tip
of
the
stimulator
arm
was
placed
just
close
to
the
receptive
field
so
that
no
force
was
generated.
Servo-­‐controlled
mechanical
stimulation
(Series
300B
dual
mode
servo
system,
Aurora
Scientific,
Canada)
was
used
to
measure
mechanosensitivity.
The
computer
controlled
ascending
series
of
square-­‐shaped
force
stimuli
was
applied
to
the
most
sensitive
spot
of
the
receptive
field
at
60-­‐s
intervals.
Since
the
neural
responses
of
cutaneous
mechanosensitive
nociceptors
to
mechanical
stimuli
are
highly
correlated
with
compressive
stress
(force)
than
compressive
strain
(
displacement),17
sustained
force-­‐controlled
stimuli
(
rise
time,
100ms;
duration
of
sustained
force
plateau,
1.9s)
were
applied.
Each
force
stimulus
was
2s
in
duration
and
started
from
zero
to
12,
32,
52,
72,
92,112,132,151,171,191,210mN.
When
an
afferent
produced
a
response
to
a
particular
force
controlled
ramp,
it
received
additional
ascending
series
of
stimuli
to
construct
stimulus
response
curve
(as
shown
in
Fig.
1).
The
total
number
of
spikes
generated
during
ascending
series
of
force
pulses
before

4. 112mN
is
compared
between
different
age
groups
(see
Fig.
1).
The
mechanical
threshold
of
units
was
determined
when
an
afferent
produced
a
response
to
a
particular
force
controlled
ramp.
Chemical
Stimulation
After
mechanical
stimulation,
chemosensitivity
was
assessed
using
modified
Krebs-­‐Henseleit
solution.
To
restrict
the
chemical
stimuli
to
the
isolated
receptive
field,
a
small
metal
ring
(
internal
diameter,
5
mm;
height,
6
mm;
volume,
0.4
ml),
which
could
seal
by
its
own
weight,
was
used.
In
some
cases,
inert
silicone
grease
was
added
to
ensure
a
waterproof
seal.
After
recording
baseline
for
5
min,
the
metal
ring
was
placed
and
the
Krebs-­‐Henseleit
solution
inside
the
ring
chamber
was
removed
and
a
chemical
cocktail,
commonly
present
in
an
inflammatory
milieu,
was
applied
to
the
receptive
field
for
60
s
with
a
temperature
of
32
°C.
The
RF
was
continuously
superfused
with
Krebs
solution
(32
°C)
before
and
after
application
of
chemical
soup.
We
compared
the
latencies
for
a
response
which
was
calculated
with
time
from
onset
of
chemical
application
to
appearance
of
two
or
more
consecutive
discharges
exceeding
the
mean
frequency
+
2
SD
of
the
background
discharge
rate
during
the
control
period
(60
s).
We
also
compared
the
mean
frequency
or
total
spikes
during
a
response
between
different
groups
(see
Fig.
2).
Following
chemicals
were
used
to
prepare
this
chemical
cocktail:
bradykinin,
histamine,
serotonin,
and
prostaglandin
E2.
The
pH
of
this
cocktail
was
normal
(7.4).
The
concentrations
are
10-­‐6
M,
these
concentrations
were
determined
according
to
past
literature
(Kessler
W
1992)
and
our
pilot
study.
Ten
min
after
chemical
application,
computer
controlled
ascending
series
of
square-­‐shaped
force
stimuli
was
applied
in
a
few
experiments.
These
data
were
used
to
compare
changes
in
the
mechanical
stimulus
response
before
and
after
chemical
soup
(see
Fig.
3).
Response
criteria
for
chemical
stimulations
When
a
fiber
fulfilled
the
following
criteria,
it
was
defined
to
be
sensitive
to
a
stimulus:
(1)
the
net
increase
in
the
discharge
rate
during
the
application
period
of
60
s
for
chemical
soup
was
more
than
0.1
imp/s
from
the
background
discharge
rate
during
the
control
period
(60
s)
immediately
before
application,
and
(2)
the
instantaneous
discharge
rate
of
two
consecutive
discharges
exceeded
the
mean
+
2
SD
of
the
background
discharge
rate.
Data
analysis
Action
potentials
collected
on
a
computer
were
analyzed
off-­‐line
with
a
template
matching
function
of
spike
2
software.
Quantitative
analysis
was
carried
out
by
counting
total
impulses
generated
in
the
stimulation
period.
In
addition,
average
discharge
frequencies
during
chemical
soup
application
were
also
counted.
Only
good
signal-­‐to-­‐noise
ratio
(>2:1)
was
considered.
Statistical
analysis

5. Results
are
expressed
as
median
with
interquartile
range
(IQR).
Averaged
response
patterns
of
afferents
are
shown
with
mean
±
SEM.
Comparisons
of
the
electrophysiological
data
between
the
young
and
the
aged
rats
were
done
using
the
Mann–Whitney
U-­‐test.
Mann–Whitney
U-­‐test
was
also
used
to
compare
baseline
(before
application
of
inflammatory
soup)
spike
numbers
induced
by
mechanical
stimulation
and
the
spike
numbers
after
inflammatory
soup.
All
tests
were
made
with
GraphPad
Prism
software,
version
5
(GraphPad,
San
Diego,
CA).
Values
of
p
<0.05
were
considered
significant.
Results
1
General
properties
of
C-­‐fibers
from
young
and
senescent
animals
127
fibers
were
identified.
86
C-­‐fiber
nociceptors
innervating
the
hairy
skin
of
rat
hindpaw
were
studied:
64
from
the
young
rats
and
22
from
aged
rats.
Conduction
velocity
was
not
different
between
two
age
groups.
The
conduction
velocities
of
the
control
C-­‐fibers
ranged
from
0.1
to
1.5
m/s
(0.54
±
0.06
m/s,
IQR:
0.1-­‐1.0m/s),
and
those
of
the
aged
C-­‐fibers
were
between
0.1
and
1.2
m/s
(0.59
±
0.09
m/s
IQR:0.18-­‐1.2m/s).
Part
of
the
reason
we
found
so
few
mechanically
sensitive
and
chemically
sensitive
C-­‐
fibers
in
aged
rats
when
compared
with
young
rats
might
have
been
a
reported
remarkably
decreased
proportion
of
mechano-­‐responsive
C-­‐fibers
and
an
notable
increase
in
the
proportion
of
mechano-­‐
insensitive
C-­‐fibers
in
aged
rats
(Taguchi
2010),
which
phenomenon
is
also
found
in
humans(Namer,
2009).
Since
we
only
identify
the
C-­‐fibers
by
using
manual
probing
with
a
glass
rod,
therefore,
we
found
much
less
mechano-­‐responsive
c-­‐fibers
in
aged
group
in
comparison
with
young
group.
There
was
no
significant
increase
in
the
discharge
rates
of
spontaneous
activity,
which
were
0.05
imp/s
(IQR:
0-­‐0.14
imp/s)
in
young
rats
and
0.02
imp/s
(IQR:
0-­‐0.10
imp/s)
in
old
rats,
respectively.
In
this
study
all
tested
C-­‐fibers
responded
to
the
inflammatory
soup
stimulation,
and
they
had
a
single
spot
like
receptive
field.
2
The
mechanical
thresholds
are
different
between
youth
and
aged
rats.
Although
the
Primary
afferent
response
to
mechanical
stimuli
of
different
age
groups
looks
the
same,
the
mechanical
thresholds
are
different
between
youth
and
aged
rats.
Mechanical
threshold
measured
by
a
train
mechanical
stimulus
in
the
aged
skin
median;
68.44
mN
(IQR:
52.1–92.1
mN),
n
=
18)
was
significantly
higher
than
that
in
the
younger
skin
(median;
52.67
mN
(IQR:
33.6–72.0
mN),
n
=
57,
p
<
0.05,
Mann–Whitney
U-­‐test).
In
addition,
the
magnitude
of
the
mechanical
response
during
the
first
6
stimulus
(from
13mN
to
112mN)
was
significantly
lower
in
the
aged
skin
(22.5
spikes
(IQR:
10.75–34.25
spikes))
than
in
the
young
(31.0spikes
(IQR:
24.25–42
spikes),
p
<
0.05,
Mann–Whitney
U-­‐test).

6. <6 and >18 mechanical threshold
t=0.03
Mech.threshold(mN)
young<6
old>18
0
50
100
150
200
112mn mech mag *
t=0.048
<
6
m
onth
>
18
m
onth
0
20
40
60
80
Figure
1
Primary
afferent
responses
to
mechanical
stimuli.
(a–g)
Digitized
oscilloscope
tracings
of
afferent
responsive
to
mechanical
stimuli.
Single
action
potentials
were
recorded
from
fine
filaments
teased
from
medial
or
lateral
plantar
nerves
of
control
mice
while
the
receptive
field
was
stimulated
by
a
feedback-­‐controlled
force
stimulator.
Seven
consecutive
recordings
show
increasing
responses
to
the
ascending
series
of
force
(h)
stimuli.
The
stimulus
duration
of
each
pulse
was
2s
and
they
were
delivered
at
30
s
intervals.
(i)
Comparison
of
mechanical
response
thresholds
between
<6
months
old
(n=57),
>18
months
(n=18)
rats.
2
Senescent
rats
have
longer
latency
in
responses
to
inflammatory
soup.
The
onset
of
neuronal
response
to
chemical
stimulation
was
significantly
delayed
in
the
afferents
from
senescent
rats.
In
the
aged
mechano-­‐responsive
C-­‐nociceptors
the
response
latency
to
inflammatory
soup
(median:
15
seconds,
(IQR:
9-­‐23
seconds))
was
significantly
longer
than
that
in
the
younger
skin
(median:
10
seconds,
(IQR:
8-­‐14
seconds))
(p
<
0.05,
Mann–Whitney
U-­‐test),
while
the
magnitude
of
the
response
was
not
different
between
the
two
age
groups.
Intensity
measured
by
total
net
spikes
in
the
aged
skin
(median:
210.5
(IQR:
157.3–281.5),
n
=
20)
was
no
different
from
that
in
the
young
skins
(median:
194.0
(IQR:
139.3-­‐319.8),
n
=
44,
p
=0.93,
Mann–Whitney
U-­‐test).
Our
observation
suggests
that
initiation
of
chemosensitivity
within
afferents
from
senescent
rats
is
slow
but
once
they
are
activated
they
can
produce
a
same
response
as
afferents
from
younger
rats,
which
were
proved
by
the
same
numbers
of
total
net
spikes
induced
by
inflammatory
soup
in
young
and
aged
rats.

7.
<6 >18new latency
P=0.018
latency(sec)
<6
m
onth
>18
m
onth
0
10
20
30
40
50
<6 month >18 month peak frequency no significan
P=0.56
peakfrequency
<6
m
onth
>18
m
onth
0
5
10
15
20
25
chemi net <6 >18
p=0.78
Totalspikes
<
6
m
onth
>
18
m
onth
0
200
400
600
Figure
2
Specimen
records
from
4
single
primary
afferents
2
months
(a),
6
months
(b),
18
months
(C)
and
26
months
(d)
rat
hairy
skin
during
trials
of
inflammatory
(mixture
of
chemicals
present
in
an
inflammatory
condition).
Ordinate:
frequency
of
discharges;
‘inflammatory
soup’
was
applied
for
1
min.
Comparison
of
latencies(e),
which
was
calculated
with
time
from
onset
of
chemical
application
to
appearance
of
clear
response.
(f)
Comparison
of
mean
frequency
(Mann-­‐Whitney
test)
(g)
total
spikes
during
a
response.
3
Inflammatory
soups
sensitize
mechanical
responses
of
both
young
and
senescent
rats.
However
young
rats
show
longer
and
stronger
sensitization.
Application
of
inflammatory
soup
had
sensitization
effect
on
mechanical
responses.
Before
and
after
inflammatory
soup
application,
a
series
of
mechanical
stimulation
were
applied
to
get
mechanical
response
curve.
Inflammatory
soup
was
super
perfused
for
1
min.
The
mechanical
stimulus
response
curves
before
and
after
inflammatory
soup
application
for
young
rats
(n=34),
and
aged
rats
(n=12)
are
respectively
shown
in
a,b.
Ordinate:
total
spikes/stimulation.
We
compared
the
spikes
number
between
the
baseline
mechanical
responses
of
nerve
fibers
and
that
of
after
inflammatory
soup
application
of
the
same
fiber.
All
numbers
were
counted
at
the
same
force
level
on
the
same
fiber
before
and
after
chemical
soup
application.
Therefore,
even
after
inflammatory
soup
application,
the
same
fiber
might
fire
at
different
threshold
force,
mostly
at
a
lower
threshold;
we
still
counted
the
number
of
spikes
that
occurred
at
the
same
force
level
as
the
thresholds
and
stimulation
intensities
indicated
by
baseline
mechanical
responses.

8. In
the
hairy
skin
preparation,
application
of
the
chemical
soup
caused
the
afferent
firing
rate
to
be
significantly
increased
during
controlled
mechanical
stimuli.
(One
sample
t-­‐test).
The
soup
enhanced
firing
rate
in
young
rats
during
controlled
mechanical
stimuli.
The
percentages
of
spikes
compared
to
baseline
in
each
phases
are:
s1:
163.4%
s2:225%;
s3:
199%;
s4:190%,
s5:181%;
s6:141%,
which
are
significantly
higher
from
s2
to
s5
compared
to
baseline
(Fig.
3).
In
aged
rats,
the
percentages
are:
91%,
88%,
132%,
184%,
161%,
and
128%
respectively.
In
aged
skin,
during
the
s1,
s2,
s3,
s5
and
s6
phase
of
controlled
stimulation
after
soup,
there
was
no
significant
change
in
activity
(Fig.
3).
Compared
to
young
animals,
the
sensitizing
effect
of
the
chemical
soup
on
the
old
animals
was
only
seen
significant
at
S4
after
soup
administration.
Also,
the
percentages
of
changes
in
firing
rates
are
different.
Compare
to
aged
skin,
the
extent
of
increases
in
s1
to
s2
in
young
skin
were
higher
(s1
P=0.047,
s2
P=0.037)
(Fig
3
c).
A
specimen
recording
showing
the
excitatory
effect
of
soup
on
afferent
nerve
activity
during
controlled
stimulation
of
a
young
and
old
rat
can
be
seen
in
Fig.
3.
Figure
3
Specimen
demonstrating
the
sensitizing
effect
of
sp
during
normal
and
stimulation
is
shown
in
fig
a,b.
Application
of
inflammatory
soup
had
sensitization
effect
on
mechanical
responses.
Before
and
after
inflammatory
soup
application,
a
series
of
mechanical
stimulation
were
applied
to
get
mechanical
response
curve.
Inflammatory
soup
was
super
perfused
for
1
min.
The
mechanical
stimulus
response
curves
before
and
after
inflammatory
soup
application
for
young
rats
(n=34),
and
aged
rats
(n=12)
are
respectively
shown
in
a,b,c.
Ordinate:
total
spikes/stimulation.
<6 mon sensitization
s1 s2 s3 s4 s5 s6
0
50
100
150
200
250
Legend
Legend
*** ***
*
***
**
*
>18 mon sensitization
s1 s2 s3 s4 s5 s6
0
50
100
150
200
250
Legend
Legend*
*
% of mech sensitization<6 >18
s1
s2
s3
s4
s5
s6
-50
0
50
100
150
<6 month
>18 month
** #
*
#
** *** *** **
*
baseline and % s2 <6 >18 m*
%ofBaseline
B
aseline
young
rats
s2
aftersoup
young
rats
s2
B
aseline
aged
rats
s2
aftersoup
aged
rats
s2
-50
0
50
100
150 **
NS
baseline and % s4 <6 >18 m*
%ofBaseline
B
aseline
young
rats
s4
aftersoup
young
rats
s4
B
aseline
aged
rats
s4
aftersoup
aged
rats
s4
0
50
100
150
*** *
Fig
3
Effect
of
inflammatory
soup
(10-­‐8
M)
on
skin
afferent
activity
in
young
(black
squares)
and
aged
(open
triangles)
rats.
Neural
activity
is
shown
in
response
to
mechanical
stimulation
of
the
skin
compared
to
pre-­‐soup
control
(represented
by
solid
line
set
at
0%).
The
sensitizing
effect
of

9. inflammatory
chemicals
was
seen
in
s1-­‐s6
in
young
skin,
although
it
is
only
seen
in
s4
in
aged
skin.
Data
are
shown
as
mean
±
SEM.
*P
<
0.05;
one
sample
t-­‐test;
n=34
fibers
for
young
rats;
n=12
fibers
for
aged
rats.
Discussion
Ageing
is
of
interest
because
ageing
influences
morphological
and
functional
features
of
cutaneous
mechanical
transducers
and
mechanosensitive
ion
channels,
sensory
innervation,
neurotransmitters
and
even
vascular
system
in
the
skin(Ageing
Res
Rev.
2014
Jan;13C:90-­‐99.Effect
of
ageing
on
tactile
transduction
processes.
Decorps
J.).
Some
age-­‐related
disappearances
in
epidermal
C-­‐fiber
endings
were
previously
reported
to
be
earlier
or
more
markedly
than
those
in
myelinated
fiber
endings
(Pare
et
al.,
2007
and
Ceballos
et
al.,
1999). The
response
to
chemical
is
of
interest
because
ageing
might
have
notable
effect
on
different
response
to
endogenous
or
exogenous
substance
such
as
bradykinin,
histamine,
and
prostaglandin.
In
addition,
there
could
be
different
sensitization
process
in
aged
compared
with
young
animals.
The
present
study
assessed
in
rats
whether
there
is
an
ageing
related
pain
sensation
change.
We
found
that
although
the
net
intensity
has
no
difference
between
young
and
senescent
rats,
aged
rats
developed
a
relative
longer
latency
in
response
to
chemical
stimulus.
In
addition,
young
rats
showed
lower
mechanical
threshold
and
stronger
mechanical
response
to
stimulation.
Also
young
rats
presented
a
stronger
sensitization
of
mechanical
response
after
chemical
stimulation
compared
to
senescent
rats.
It
is
generally
agreed
that
the
cool
and
warm
detection
thresholds
assess
the
function
of
small
myelinated
Aδ
fibres
and
unmyelinated
C
fibres,
whereas
sensitivities
to
vibration
and
tactile
stimulation
assess
the
function
of
large
myelinated
fibres,
respectively
(Campero
et
al.,
1996
and
Verdugo
and
Ochoa,
1992).
Abnormalities
of
the
sensory
system,
such
as
detection
thresholds,
nerve
conduction
velocities,
structural
changes
of
sensory
fibres
can
also
develop
because
of
ageing.
For
example,
modest
functional
abnormality
of
small
sensory
fibres
was
shown
in
the
older
subjects,
who
displayed
increased
warm
detection
threshold
compared
to
young
adults
(Fromy
et
al.,
2010).
Also
the
degree
of
activity-­‐
dependent
conduction
velocity
slowing
in
response
to
high
frequency
stimulation
was
more
pronounced
in
aged
subjects
(Namer,
2009).
These
changes
in
the
axonal
properties
of
C-­‐fibres
in
aged
subjects
are
compatible
with
hypoexcitability
of
the
fibers.
Decreased
mechanical
response
It
was
suggested
that
the
ratio
of
mechano-­‐responsive
fibres
to
mechano-­‐insensitive
fibres
was
shifted
in
favor
of
the
mechano-­‐insensitive
fibres
in
older
subjects
(Namer
et
al.,
2009
and
Orstavik
et
al.,
2006,
Taguchi
2010
pain).
However,
since
we
used
the
probe
stimulation
to
identify
only
mechano-­‐responsive
C-­‐fibers
instead
of
electrically
identifying
both
mechano-­‐responsive
and
mechano-­‐insensitive
C-­‐fiber
population,
we
did
not
see
such
a
ratio
shifting.
But
we
found
much
less
mechano-­‐responsive
fibers
(22)
in
our
aged
group
compared
with
young
rats
(64)
which
might
partially
be
explained
by
the
ratio
shifting
from
mechano-­‐responsive
dominant
fibres
to
mechano-­‐insensitive
fibres.

10. Our
results
showed
a
higher
mechanical
threshold
of
response
in
the
aged
group
in
comparison
to
young
rats,
which
is
well
in
line
with
the
previous
observation
in
SD
rats
(Taguchi
2010
pain).
The
mechanical
response
of
individual
mechano-­‐responsive
c
fibres
tends
to
decrease
with
age.
This
may
resulted
from
following
reasons:
First,
the
ageing
effects
on
the
structure
and
function
of
these
mechanosensitive
ion
channels
could
contribute
to
the
age-­‐related
mechano-­‐
response.
Activation
of
mechanosensitive
ion
channels
is
important
for
the
detection
of
mechanical
stimuli
required
for
transduction
to
electrical
signals
in
sensory
neurons.
Expression
of
sodium
channel
Nav1.8
and
TRPV1
expression
has
been
shown
to
be
lowered
in
cutaneous
nerves
of
aged
mice
(Wang
s,
neurobiol
Aging,
2006)
and
is
related
to
reduced
thermal
sensitivity.
The
GFRalpha3
receptor,
which
binds
the
growth
factor
artemin
and
is
expressed
by
TRPV1-­‐positive
neurons,
was
also
decreased
in
the
DRG
of
aged
animals.
These
findings
indicate
that
loss
of
thermal
sensitivity
in
aging
animals
may
result
from
a
decreased
level
of
TRPV1
and
Nav1.8
and
decreased
trophic
support
that
inhibits
efficient
transport
of
channel
proteins
to
peripheral
afferents.
Beside,
some
findings
have
shown
that
selective
TRPV1
antagonists
cause
a
reduction
in
both
thermal
and
mechanical
hyperalgesia
and
TRPV1
also
plays
a
role
in
mechanical
hyperalgesia
(Pomonis
et
al.,
2003;
Walker
et
al.,
2003;
Tang
et
al.,
2007;
Btesh
J,
2013).
ASIC
3channel
has
also
been
shown
to
detect
some
cutaneous
touch
and
painful
stimuli
(Fromy,
2012).
Other
ion
channels
such
as
TRPA1,
MEC4/MEC-­‐10
and
two-­‐pore
domain
potassium
(K+)-­‐selective
channels
(such
as
TREK1
and
TRAAK)
might
also
be
playing
as
a
neuronal
mechanosensitive
channel
(Decorps
J,
2014).
Although
the
ageing
effects
on
the
structure
and/or
the
function
of
these
mechanosensitive
ion
channels
are
not
described,
one
can
speculate
that
they
could
contribute
to
the
age-­‐related
tactile
defect.
Second,
changes
in
the
physical
properties
of
aged
skin
may
influence
the
nociceptor
response.
There
are
pronounced
age-­‐induced
changes
in
the
viscoelastic
properties
of
the
skin
and
underlying
tissue.
Profound
differences
in
some
mechanical
properties
of
the
skin
were
found
between
young
and
adult
rats.
The
compliance
of
the
skin
is
decreased
in
adult
rats
when
compared
with
young
rats18
(Baumann
KI,
Hamann
W,
Leung
MS:
Mechanical
properties
of
skin
and
responsiveness
of
slowly
adapting
type
I
mechanoreceptors
in
rats
at
different
ages.
J
Physiol
1986;
371:
329-­‐37)
During
rats’
adulthood,
there
was
a
subsequent
tortuosity
of
the
distorted
elastic
fibers
which
have
lost
their
original
elasticity
and
interlock
with
the
collagen
bundles.
Interlocking
of
both
collagen
and
elastic
fibers
decrease
tissue
compliance19
(Imayama
S.
Am
J
Pathol
1989).
In
human
being,
the
thickness
of
the
dermis
also
decreases
with
age
and
this
is
accompanied
by
a
decrease
in
number
of
mast
cells
and
fibroblasts,
and
a
decrease
in
the
generation
of
collagen,
elastin,
glycosaminoglycans,
and
hyaluronic
acid.
It
is
thought
that
changes
in
the
amount
of
collagen,
alterations
in
tissue
reactive
oxygen
species
or
decreases
in
the
amount
of
fibroblast-­‐collagen
linkage
may
result
in
a
diminished
ability
of
the
skin
to
detect
or
propagate
mechanical
stimuli;
however,
it
has
not
yet
been
investigated.
20
(Wu
M:
Effect
of
aging
on
cellular
mechanotransduction.
Ageing
Res
Rev
2011).
We
also
found
that
in
aged
rats,
the
number
of
impulses
(magnitude
of
response)
induced
by
mechanical
stimulation
tend
to
decrease
compared
to
young
rats,
which
could
be
due
to
the
following
reasons:
First,
since
there
are
decreased
expression
of
Nav1.8
and
TRPV1
protein
in
cutaneous
nerves

11. of
aged
mice
(Wang
s,
neurobiol
Aging,
2006).
It
has
been
indicated
that
Nav1.8
sodium
channels
contribute
substantially
to
action
potential
electrogenesis
in
DRG
neurons
(J
Neurophysiol.
2001,
Renganathan).
It
is
possible
that
the
age-­‐related
expression
of
Nav1.8
could
lead
to
changes
in
less
action
potential
electrogenesis
in
aged
rats.
Secondly,
a
decreased
sodium-­‐
potassium
pump
activity
in
dorsal
root
in
aged
mice
was
observed
(Robertson,
1993).
As
it
has
been
suggested
this
decreased
basal
level
of
pump
activity
would
lead
to
relatively
depolarized
membrane
potential
and
higher
proportion
of
inactivated
sodium
channels,
which
would
result
in
hypoexcitability
of
fires
to
sensory
stimuli
(Namer
2009).
This
could
also
leads
to
fewer
spikes
to
mechanical
stimulation
in
aged
skin.
Chemical
responses
and
sensitized
mechanical
response
after
chemical
soup
Although
there
was
no
difference
between
young
and
aged
rats
with
the
net
spikes
induced
by
chemical
stimulation,
activities
of
nociceptors
in
response
to
chemicals
(bradykinin,
histamine,
serotonin,
and
prostaglandin
E2)
have
changed
with
ageing
shown
by
a
longer
latency
in
the
aged
rats.
Our
finding
is
supported
by
previous
report
that
latency
of
mechanoresponsive
C
fibers
to
10uM
bradykinin
was
significantly
longer
in
the
aged
SD
rats
(Taguchi,
2010).
Also,
our
results
showed
that
after
chemical
soup
the
mechanical
responses
are
enhanced
both
in
young
and
old
rats.
Previous
report
showed
that
local
application
of
SP
had
a
sensitizing
effect
on
joint
afferents
in
response
to
movements
in
old
animals
(McDougall
JJ,
2007).
Here,
our
results
first
time
showed
that
this
sensitization
was
more
prominent
in
young
rats
than
old
rats,
which
was
evidenced
by
stronger
enhanced
mechanical
responses
in
young
rats.
We
found
that
percentages
of
changes
in
firing
rates
induced
by
inflammatory
soup
were
higher
in
young
rats
than
in
aged
rats.
Also
the
increased
firing
could
be
seen
in
all
mechanical
stimulation
phases
including
s1
to
s6,
where
in
aged
rats,
it
was
only
seen
in
s4.
One
reason
for
a
longer
latency
of
inflammatory
mediator
induced
response
and
weakened
sensitization
in
senescent
skin
might
result
from
the
reduced
expressions
of
receptor
molecules
and
transducers
such
as
TRPV1,
bradykinin
receptors,
histamine
receptors
and
serotonin
receptors,
prostaglandin
receptors.
Indeed,
in
rat
spinal
cord,
study
using
quantitative
immunohistochemistry
for
serotonin
(5-­‐HT)
and
tyrosine
hydroxylase
(TH)
in
male
Wistar
rats
of
3
and
24
months
revealed
significant
age-­‐associated
declines
in
the
monoaminergic
innervation
(Ranson,
R.
N.,2003,
Age-­‐associated
changes
in
the
monoaminergic
innervation
of
rat
lumbosacral
spinal
cord.
Brain
Res).
In
the
dorsal
root
ganglia
of
aged
rats,
SP-­‐like
immunoreactivity
significantly
reduced
compared
to
young
adults
(Bergman,
1996).
Although
there
are
no
study
available
as
for
the
age-­‐related
changes
of
bradykinin,
serotonin
and
prostaglandin
E2
expressions
in
aged
rats,
it
has
been
shown
that
TRPV1
expression
in
peripheral
nerve
is
lower
in
aged
mice
(Wang
s,
neurobiol
Aging,
2006).
This
created
a
possibility
that
reduced
TRPV1
expression
with
ageing
might
lead
to
decreased
bradykinin-­‐evoked
and
prostaglandin-­‐evoked
nociceptor
excitation
and
bradykinin-­‐induced
mechanical
hyperalgesia.
Bradykinin
is
produced
in
response
to
tissue
injury,
inflammation,
or
ischemia
and
binds
to
PLC
coupled
(BK2)
receptors
on
sensory
neurons
(McMahon
et
al.,
2006).
Bradykinin
elicits
acute
pain
through
immediate
excitation
of
nociceptors,
followed
by
a
longer
lasting
sensitization
to
thermal
and

12. mechanical
stimuli
(Dray
and
Perkins,
1993).
Genetic
and
electrophysiological
studies
suggest
that
bradykinin-­‐evoked
thermal
hypersensitivity
is
produced
through
PLC-­‐mediated
potentiation
of
TRPV1
(Cesare
et
al.,
1999;
Chuang
et
al.,
2001;
Premkumar
and
Ahern,
2000).
Several
studies
have
suggested
that
TRPV1
is
essential
to
the
BK-­‐evoked
responses
(Shin
et
al.,
2002;
Ferreira
et
al.,
2004,
Neurosci
Res.
2008
Katanosaka
K).
In
addition,
histamine-­‐dependent
itch
is
mediated
by
a
subset
of
C-­‐fiber
afferents
that
express
TRPV1
and
the
histamine
receptor
(Shim
WS,
2007.
TRPV1
mediates
histamine-­‐induced
itching
via
the
activation
of
phospholipase
A2
and
12-­‐lipoxygenase.
J.
Neurosci.).
Prostaglandins
(PGs),
another
class
of
fatty
acid
derivatives,
are
produced
at
sites
of
inflammation
and
mediate
inflammatory
responses
and
sensitization
by
a
variety
of
mechanisms.
Protein
kinase
C
(PKC)
and
PKA
downstream
of
prostaglandin
E2
receptors,
sensitize/activate
multiple
molecules
including
transient
receptor
potential
vanilloid-­‐1
(TRPV1)
channels,
purinergic
P2X3
receptors,
and
voltage-­‐gated
calcium
or
sodium
channels
in
nociceptors,
leading
to
hyperalgesia
(Biol
Pharm
Bull.
2011,Prostaglandin
E2
and
pain-­‐-­‐an
update.
Kawabata
A).
Recently
it
was
shown
that
inflammatory
mediators
such
as
prostaglandin-­‐E2
or
bradykinin
cause
hyperalgesia
by
activating
cellular
kinases
that
phosphorylate
TRPV1,
a
process
that
relies
on
a
scaffolding
protein,
AKAP79,
to
target
the
kinases
to
TRPV1(J
Neurosci.
Btesh
J,
2013).
We
speculated
that
reduced
TRPV1
expression
with
ageing
could
lead
to
reduced
bradykinin-­‐evoked
and
prostaglandin-­‐
evoked
nociceptor
excitation
and
bradykinin-­‐induced
mechanical
hyperalgesia.
Also
the
histamine
induced
C-­‐fiber
excitation
might
decrease
with
aging
since
TRPV1
expressions
are
decreased
with
aging.
One
can
speculate
that
the
ageing
effects
on
the
structure
of
other
ion
channels
such
as
TRPA1,
could
contribute
to
the
age-­‐related
chemical
responses.
Interestingly,
a
study
showed
that
the
mechanosensitivity
of
mouse
colon
afferent
fibers
and
their
sensitization
by
inflammatory
mediators
require
TRPV1
and
ASIC
3
(J
Neurosci.
2005
Jones
RC
3rd).
And
combined
genetic
and
pharmacological
inhibition
of
TRPV1
and
P2X3
attenuates
colorectal
hypersensitivity
and
afferent
sensitization
by
inflammatory
soup
was
also
significantly
attenuated
(Kiyatkin
ME,
2013).However,
whether
this
also
applied
to
aged
cutaneous
afferents
needs
to
be
investigated
in
the
future.
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