Stress and cortisol – the puzzle of their influence on pain sensitivity

fernand.anton@uni.lu

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!!!