1. Stress and cortisol – the puzzle of their influence on pain sensitivity fernand.anton@uni.lu

2. Introduction Stress may not only lead to the well known phenomenon of stress-induced suppression of pain (SIA) It may under certain conditions also induce an enhancement and prolongation of pain states Several cascades may be involved We have been focusing on the impact of a relative hypocortisolism

3. Animal studies on inflammatory pain Preclinical and experimental studies in humans

4. Animal studies: genetically different strains of rats (Fischer and Lewis) animals with pharmacologically manipulated HPA-axis reactivity rats exposed to stress

5. Material & methods Hargreaves & von Frey tests Paw 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

6. 1 st 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 7  On day 0, intraplantar injection of carrageenan 2.7% (left hindpaw) and NaCl 0.9% (right hindpaw) Sacrifice -201 4 7 Time (d) Lewis rats: low level of corticosterone Fischer rats: high level of CORT.

7. Comparison of pain behavior and paw volume in 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) 01247 Δ paw volume (% control) 100 120 140 160 180 200 Paw volume *** time (day) 01247 Δ PWL (% control) 20 40 60 80 100 120 Plantar test ** *** time (day) 01247 Δ Force (% control) 0 20 40 60 80 100 120 von Frey test Fischer rats (n=10): ■ Lewis rats (n=10): ■

8. 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 Density of Ox-42 (%left/right side) *** L5L6 0 50 100 150 200 250 300 Level 200 250 ns Level L5L6 Density of GFAP (%left/right side) 0 50 100 150 *** L5L6 Δ number of FosB/ΔFosB-ir cells (left-right side) 0 20 40 60 80 Level FosB/ΔFosB Microglia Astrocytes

9. Discussion inflammation 0 1247 - ↑ 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, PGE 2, AP-1 complex Maintenance of pain hypersensitivity Substance P Microglial activation Better recovery in FIS Pain thresholds lower in LEW microglial expression higher in LEW - GC - (Goulding et al., 1998)

10. 2 nd study: effects of carrageenan- induced inflammation after pharmacological manipulation of the HPA axis

11. 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 1 Short term inflammation Animal model: Wistar rats Long term inflammation Animal 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 -201 4 7 10 14 Time (d)

12. Effect of GRantagonist/agonist treatment on pain behaviors after an acute inflammation time (day) pre treat.pre inf.1 Δ paw withdrawal latency (% control) 20 40 60 80 100 *** Plantar test time (day) pre treat.pre inf. 1 Δ paw volume (% control) 80 100 120 140 160 *** Paw volume time (day) pre treat.pre inf.1 Δ force (% control) 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): ■

13. 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 L5L6 Number of FosB/ΔFosB-ir cells (left-right side) 0 2 4 6 8 10 12 14 16 18 *** TNFα level L5L6 Number of TNFα-ir cells (left-right side) 0 20 40 60 80 100 120 *** Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■

14. 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 level L5L6 Density of GFAP (% left/right side) 0 100 200 300 400 mGluR5 level L5L6 Density of mGluR5 (% left/right side) 0 50 100 150 200 250 300 *** * ** *** Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■

15. 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): ■ *** Paw volume Δ force (% control) time (day) pre treat. pre inf. 1471014 0 20 40 60 80 100 *** von Frey test

16. 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 level L5L6 Number of FosB/ΔFosB-ir cells (left-right side) 0 20 40 60 80 100 120 140 *** TNFα level L5L6 Number of TNFα-ir cells (left-right side) 0 20 40 60 80 100 120 140 160 180 ***

17. Effect of GRantagonist/agonist treatment on spinal cell activation after a chronic inflammation astrocytes level L5L6 Density of GFAP (% left/right side) 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 L5L6 Density of mGluR5 (% left/right side) 0 100 200 300 400 ** * *

18. 3 rd study: effects of carrageenan- induced inflammation after a chronic restraint stress

19. 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 -201 4 7 10 14 Time (d) -7 Animal model: Wistar rats

20. Effect of chronic restraint stress on behavioural tests after a chronic inflammation Paw volume Time (day) Pre stress Pre Inf. 1471014 paw volume (% control) 80 100 120 140 160 180 * *** Plantar test Time (day) Pre stress Pre Inf. 1471014 PWL (% control) 30 40 50 60 70 80 90 100 *** von frey test Time (day) Pre stress Pre Inf. 1471014 Force (% control) 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

21. 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 L5L6 Number of FosB/ΔFosB-ir cells (left-right side) 0 10 20 30 40 *** TNFα Level L5L6 Number of TNFα-ir cells (left-right side) 0 20 40 60 80 ***

22. 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 Level L5L6 Density of GFAP (% left/right side) 0 50 100 150 200 250 300 *** microglia Level L5L6 Density of Iba1 (% left/right side) 0 50 100 150 200 250 300 *** Density of mGluR5 Level L5L6 Density of mGluR5 (% left/right side) 0 50 100 150 200 250 300 350 *** Stress (n=10): ■ Control (n=10): ■

23. 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 hypernociception Stress ↑ 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)

24. 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.

25. Studies in humans: prospective preclinical studies experimental psychophysical studies

26. Clinical

27. Cooperation with the colleagues from the Omsk State Medical Academy

28. Psychophysics: Causal relationships: Do alterations of glucocorticoid levels have an impact or pain sensitivity or....?

30. Protocol of the study

31. Saliva and plasma concentrations of cortisol and ACTH

32. Lowering of mechanical pain thresholds (higher pain sensitivity) under conditions of relative hypocortisolosm

33. Relative hypocortisolism depresses mechanically induced wind-up

34. Relative hypocortisolism enhances repeated interdigital web pinching-induced hyperalgesia

35. (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

36. 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??

37. 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......

39. Thanks for your attention!!!