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Stress and cortisol – the puzzle of their influence on pain sensitivity
Introduction
Stress may not only lead to the well known phenomenon ofstress-induced suppression of pain (SIA)
It may under certain conditions also induce an enhancementand prolongation of pain states
Several cascades may be involved
We have been focusing on the impact of a relative hypocortisolism
Material & methods
Hargreaves & von Frey testsPaw volume
Blood sampling
Post mortem brains, DRG and spinal cord (L5+L6) sampling
Blood sampling → Cort. Assay
Inflammation → carrageenan 2,7%
Sacrifice → intracardial perfusion of zamboni’s fixative
Spinal cord → IHC (DAB method) for FosB/ΔFosB, TNFα, GFAP, OX-42/Iba1, mGluR5
DRG → immunofluorescence
1st study: effects of carrageenan-induced in Lweis and Fischer rats
synopsis
Pre-inflammation(d -2, -1, & 0)
Behavioural tests (Hargreaves & von Frey) and paw volume on day -2, -1, 0 and 1,2, 4 and 7On day 0, intraplantar injection of carrageenan 2.7% (left hindpaw) and NaCl 0.9% (right hindpaw)
Sacrifice
-2 0 1 4 7
Time (d)
Lewis rats: low level of corticosteroneFischer rats: high level of CORT.
Comparison of pain behavior and paw volumein Lewis & Fischer rats
Data are shown as mean + SD. ** P<1%; ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison Fis vs Lew
******
***
time (day)0 1 2 4 7
Δ p
aw v
olu
me
(% c
on
tro
l)
100
120
140
160
180
200
Paw volume
***
***
time (day)
0 1 2 4 7
Δ P
WL
(%
co
ntr
ol)
20
40
60
80
100
120
Plantar test**
***
***
time (day)0 1 2 4 7
Δ F
orc
e (%
co
ntr
ol)
0
20
40
60
80
100
120
von Frey test
Fischer rats (n=10): ■ Lewis rats (n=10): ■
Comparison of neuron and glia cell activation in Lewis and Fischer rats
Fischer rats (n=10): ■ Lewis rats (n=10): ■
Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison Lew vs Fis
De
ns
ity
of
Ox
-42
(%le
ft/r
igh
t s
ide
)
******
L5 L60
50
100
150
200
250
300
Level
200
250ns ns
LevelL5 L6
De
ns
ity
of
GF
AP
(%le
ft/r
igh
t s
ide
)
0
50
100
150
*** ***
L5 L6
Δ n
um
be
r o
f F
os
B/Δ
Fo
sB
-ir
ce
lls
(le
ft-r
igh
t s
ide
)
0
20
40
60
80
Level
FosB/ΔFosB
Microglia Astrocytes
Discussioninflammation
0 1 2 4 7
- ↑ thermal and mechanical hypersensitivity and paw volume- ↑ expression FosB/ΔFosB
Central and peripheral sensitization
Activation of neutrophils(Fecho & Valtschanoff 2006)
GC+
Fos expression, responses to behavioral tests, and paw volume higher in Fischer
Pro-inflammatory cytokines, NF-κB, BK, PGE2, AP-1 complex
Maintenance of pain hypersensitivity
Substance P
Microglial activation
Better recovery in FISPain thresholds lower in LEW
microglial expression higher in LEW
-GC-(Goulding et al., 1998)
2nd study: effects of carrageenan-induced inflammation after
pharmacological manipulation of the HPA axis
synopsis
Daily (d-2 to d0) subcutaneous injection of RU 486 (20 mg/Kg/d), dexamethasone (5 mg/Kg/d) or vehicle (olive oil + 1% ethanol). Behavioural tests (Hargreaves & von Frey) and paw volume on day -5, -4, -3, -2, -1, 0 and 1.On day 0, intraplantar injection of carrageenan 2.7% (left hindpaw) and NaCl 0.9% (right hindpaw)
Time (d)
Pre-treatment(d-5, -4, -3)
Pre-inflammation(d-2, -1, 0) Sacrifice
-5 -4 -3 -2 -1 0 1Short term inflammationAnimal model: Wistar rats
Long term inflammationAnimal model: Wistar rats
Pre-treatment(d -5, -4 & -3)
Pre-inflammation(d -2, -1, & 0)
Daily (d-2 to d14) subcutaneous injection of RU 486 (4 mg/Kg/d), dexamethasone (0.5 mg/Kg/d) or vehicle (olive oil + 1% ethanol). Behavioural tests (Hargreaves & von Frey) and paw volume on day -5, -4, -3, -2, -1, 0 and 1, 4, 7, 10 and 14.On day 0, intraplantar injection of carrageenan 2.7% (left hindpaw) and NaCl 0.9% (right hindpaw)
Sacrifice
-5 -2 0 1 4 7 10 14
Time (d)
Effect of GRantagonist/agonist treatment on pain behaviors after an acute inflammation
time (day)pre treat. pre inf. 1
Δ p
aw
wit
hd
raw
al
late
nc
y(%
co
ntr
ol)
20
40
60
80
100
***
***
Plantar test
time (day)
pre treat. pre inf. 1
Δ p
aw
vo
lum
e (
% c
on
tro
l)
80
100
120
140
160***
***
Paw volume
time (day)pre treat. pre inf. 1
Δ f
orc
e (
% c
on
tro
l)
0
20
40
60
80
100
***
***
von Frey test
Data are shown as mean + SD. ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison treatment vs control
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
Effect of GRantagonist/agonist treatment on spinal cell activation after an acute inflammation
Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison treatment vs control
FosB/ΔFosB
level
L5 L6
Nu
mb
er
of
Fo
sB
/ΔF
os
B-i
r c
ell
s(l
eft
-rig
ht
sid
e)
0
2
4
6
8
10
12
14
16
18
***
***
***
***
TNFα
level
L5 L6Nu
mb
er
of
TN
Fα
-ir
ce
lls
(le
ft-r
igh
t s
ide
)
0
20
40
60
80
100
120
***
***
***
***
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
Effect of GRantagonist/agonist treatment on spinal cell activation after an acute inflammation
Data are shown as mean + SD. *P<5%, ***P<0.1%, t-test 2-tailed; comparison treatment vs control
Astrocytes
levelL5 L6
De
ns
ity
of
GF
AP
(%
le
ft/r
igh
t s
ide
)
0
100
200
300
400 mGluR5
level
L5 L6
De
ns
ity
of
mG
luR
5 (
% l
eft
/rig
ht
sid
e)
0
50
100
150
200
250
300
*** ***
*
*** **
******
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
Effect of GRantagonist/agonist treatment on behavioural tests after a chronic inflammation
Data are shown as mean + SD. ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison treatment vs control
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
time (day)
pretreat.
pre inf.
1 4 7 10 14
Δ p
aw
vo
lum
e
(% c
on
tro
l)
80
100
120
140
160
180
200
***
******
***
***
*** ***
*** ***
***
Paw volume
time (day)pretreat.
pre inf
1 4 7 10 14
Δ p
aw
wit
hd
raw
al
late
nc
y
(% c
on
tro
l)
20
40
60
80
100
***
******
*** ***
******
***
***
***
Plantar test
Δ f
orc
e
(% c
on
tro
l)
time (day)
pretreat.
preinf.
1 4 7 10 14
0
20
40
60
80
100
***
***
***
***
***
*** *** ***
******
von Frey test
Effect of GRantagonist/agonist treatment on spinal cell activation after a chronic inflammation
Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison treatment vs control
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
FosB/ΔFosB
levelL5 L6
Nu
mb
er
of
Fo
sB
/ΔF
os
B-i
r c
ell
s(l
eft
-rig
ht
sid
e)
0
20
40
60
80
100
120
140
***
***
***
***
TNFα
levelL5 L6
Nu
mb
er
of
TN
Fα
-ir
ce
lls
(le
ft-r
igh
t s
ide
)
0
20
40
60
80
100
120
140
160
180
***
***
***
***
Effect of GRantagonist/agonist treatment on spinal cell activation after a chronic inflammation
astrocytes
level
L5 L6
De
ns
ity
of
GF
AP
(%
le
ft/r
igh
t s
ide
)
0
50
100
150
200
250
300
Data are shown as mean + SD. * P<5%, **P<1%, t-test 2-tailed; comparison treatment vs control
Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■
mGluR5
level
L5 L6D
en
sit
y o
f m
Glu
R5
(%
le
ft/r
igh
t s
ide
)
0
100
200
300
400
****
* *
**
3rd study: effects of carrageenan-induced inflammation after a chronic restraint stress
Synopsis
Pre-stress(d -10, -9 & -8)
Pre-inflammation(d -2, -1, & 0)
Daily (d-7 to d14) chronic restraint stress 2h/d Behavioural tests (Hargreaves & von Frey) and paw volume on day -10, -9, -8, -2, -1, 0 and 1, 4, 7, 10 and 14.On day 0, intraplantar injection of carrageenan 2.7% (left hindpaw) and NaCl 0.9% (right hindpaw)
Sacrifice
-2 0 1 4 7 10 14
Time (d)
-7
Animal model: Wistar rats
Effect of chronic restraint stress on behavioural tests after a chronic inflammation
Paw volumeTime (day)
Prestress
PreInf.
1 4 7 10 14
pa
w v
olu
me
(%
co
ntr
ol)
80
100
120
140
160
180
* ******
Plantar testTime (day)
Pre stress
Pre Inf.
1 4 7 10 14
PW
L (
% c
on
tro
l)
30
40
50
60
70
80
90
100 ******
***
***
***
von frey testTime (day)
Prestress
PreInf.
1 4 7 10 14
Fo
rce
(%
co
ntr
ol)
30
40
50
60
70
80
90
100
***
******
***
***
Stress (n=10): ■ Control (n=10): ■
Data are shown as mean + SD. * P<5%, ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison stress vs control
Effect of chronic restraint stress on spinal cell activation after a chronic inflammation
Stress (n=10): ■ Control (n=10): ■
Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison stress vs control
FosB/ΔFosB
Level
L5 L6
Nu
mb
er
of
Fo
sB
/ΔF
os
B-i
r c
ell
s(l
eft
-rig
ht
sid
e)
0
10
20
30
40
*** ***
TNFα
Level
L5 L6Nu
mb
er
of
TN
Fα
-ir
ce
lls
(le
ft-r
igh
t s
ide
)0
20
40
60
80
*** ***
Effect of chronic restraint stress on spinal cell activation after a chronic inflammation
Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison stress vs control
astrocytes
LevelL5 L6
De
ns
ity
of
GF
AP
(% l
eft
/rig
ht
sid
e)
0
50
100
150
200
250
300
*** ***
microglia
LevelL5 L6
De
ns
ity
of
Iba
1(%
le
ft/r
igh
t s
ide
)
0
50
100
150
200
250
300
******
Density of mGluR5
LevelL5 L6
De
ns
ity
of
mG
luR
5(%
le
ft/r
igh
t s
ide
)
0
50
100
150
200
250
300
350
*** ***Stress (n=10): ■ Control (n=10): ■
Discussion
Stress ↑ opioid activity Pain inhibition (Gamaro et al., 1998 Machelska et al., 2003)
Early phase of inflammation
↓ opioid activity(Dantas et al., 2005)
↑ pain sensitivity
Inflammation-induced hypernociceptionStress ↑ FosB (Perrotti et al., 2004), mGluR5 (Chaouloff et al., 2007) and TNFα expression (Frick et al., 2008), astroglial and microglial activation (Kwon et al., 2008).
Late phase of inflammation
OR: Descending inhibitory pathway? (Watkins et al., 1982)
Conclusions
Biphasic effect of the corticosterone level in Lewis and Fischer rats. (neutrophil activation followed by domination of immunosuppressive effects of glucocorticoids).
Treatment with dexamethasone inhibited pain processing, TNFα and Fos activation and increased mGluR5 expression. RU486 increased hypernociception, FosB and TNFα expression via an inhibition of the trans-repression of the pro-inflammatory genes.
Chronic stress reduced pain sensitivity during the first days of inflammation (opioid activation) and then induced a strong maintenance of hypernociception, an increased of FosB, TNFα, mGluR5 and glia cell activation.
Clinical
Cooperation with the colleagues from the Omsk State Medical Academy
Psychophysics:
Causal relationships: Do alterations of glucocorticoid levels havean impact or pain sensitivity or....?
Protocol of the study
Saliva and plasma concentrations of cortisol and ACTH
Lowering of mechanical pain thresholds (higher pain sensitivity) underconditions of relative hypocortisolosm
Relative hypocortisolism depresses mechanically induced wind-up
Relative hypocortisolism enhances repeated interdigital web pinching-inducedhyperalgesia
(First) conclusions
Our studies support the enhancement of pain processing under conditions of (stress-related) relative hypocortisolism, at least for conditions of inflammation-related pain
A disinhibition of the release of inflammatory mediators by immunocompetent and glia cells may be involved and lead to central states of sensitization
However!!!!!!
(back to the puzzle indicated in the title)
• Contrary to our studies, enhanced glucocrticoid levels have been shown to increase spinal nociceptive processing, mainly via a direct positive interaction with NMDA-receptors and a downregulation of glial glutamate transporters (This has mainly been shown for neuropathic pain: Alexander et al, 2009; Wang et al, 2004, 2005, 2006)
• In the periphery, glucocorticoids may (in synergy with sympathetic activity) lead to an ongoing sensitization ( and hence enhanced pain processing) of nociceptive nerve endings (Khasar et al, 2008; Rechling and Levine, 2009)
Are there differences between inflammatory and neuropathic pain? Do time scales play a role??
Additional potential factors and mechanisms that need consideration
• possible involvement of stress-related alterations of neurosteroid levels or reactivity (cooperation with Pierrick Poisbeau and Pascal Darbon, Strasbourg)
• stress-induced switching of descending pain modulation pathways, from inhibition to facilitation (Martenson et al, 2009; Roberts et al, 2009)
• many other factors (genes, gender and sex hormones, peri-natal experiences......
Thanks for your attention!!!