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

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. Animalstudies on inflammatorypain Preclinical and experimental studies in humans

4. Animalstudies: • genetically different strains of rats (Fischer • and Lewis) • animalswithpharmacologicallymanipulated • HPA-axisreactivity • ratsexposed to stress

5. Material & methods 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 Post mortem brains, DRG and spinal cord (L5+L6) sampling Blood sampling Hargreaves & von Frey tests Paw volume

6. 1ststudy: effects of carrageenan-induced in Lweis and Fischer ratssynopsis Lewis rats: low level of corticosterone Fischer rats: high level of CORT. Sacrifice Pre-inflammation (d -2, -1, & 0) -2 0 1 7 4 Time (d) • 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)

7. 200 *** *** 180 160 *** Δpaw volume (% control) 140 120 100 0 1 2 4 7 time (day) 120 120 100 100 *** *** 80 80 *** ΔForce (% control) 60 Δ PWL (% control) 60 40 *** Plantar test von Frey test 40 20 ** 0 20 0 1 2 4 7 0 1 2 4 7 time (day) time (day) Comparison of pain behavior and paw volumein Lewis & Fischer rats Fischer rats (n=10): ■ Lewis rats (n=10): ■ Paw volume Data are shown as mean + SD. ** P<1%; ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison Fis vs Lew

8. FosB/ΔFosB *** *** 80 60 Δnumber of FosB/ΔFosB-ir cells (left-right side) 40 20 0 L5 L6 Level *** *** 250 300 ns ns 200 250 200 150 Density of GFAP (%left/right side) Density of Ox-42 (%left/right side) 150 100 100 50 50 0 0 L5 L6 L5 L6 Level Level Comparison of neuron and glia cell activation in Lewis and Fischer rats Fischer rats (n=10): ■ Lewis rats (n=10): ■ Astrocytes Microglia Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison Lew vs Fis

9. GC - - + GC (Goulding et al., 1998) Better recovery in FIS Pain thresholds lower in LEW Fos expression, responses to behavioral tests, and paw volume higher in Fischer microglial expression higher in LEW Discussion inflammation Pro-inflammatory cytokines, NF-κB, BK, PGE2, AP-1 complex Substance P Central and peripheral sensitization Activation of neutrophils (Fecho & Valtschanoff 2006) Maintenance of pain hypersensitivity - ↑ thermal and mechanical hypersensitivity and paw volume - ↑ expression FosB/ΔFosB Microglial activation 0 1 2 4 7

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

11. Pre-treatment (d-5, -4, -3) Pre-inflammation (d-2, -1, 0) Sacrifice -5 -4 -3 -2 -1 0 1 Time (d) • 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) Sacrifice Pre-inflammation (d -2, -1, & 0) -2 0 1 7 -5 4 10 14 Time (d) Pre-treatment (d -5, -4 & -3) • 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) synopsis Short term inflammation Animal model: Wistar rats Long term inflammation Animal model: Wistar rats

12. *** Paw volume 160 140 Δpaw volume (% control) 120 *** 100 80 1 pre treat. pre inf. time (day) von Frey test Plantar test 100 *** 100 80 *** 60 80 Δforce (% control) Δpaw withdrawal latency (% control) 40 60 20 *** 40 *** 0 pre treat. pre inf. 1 20 time (day) pre treat. pre inf. 1 time (day) Effect of GRantagonist/agonist treatment on pain behaviors after an acute inflammation Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ Data are shown as mean + SD. ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison treatment vs control

13. FosB/ΔFosB TNFα 18 *** 120 *** *** 16 *** 100 14 12 80 10 Number of FosB/ΔFosB-ir cells (left-right side) Number of TNFα-ir cells (left-right side) 60 8 6 40 4 *** *** *** *** 20 2 0 0 L5 L6 L5 L6 level level Effect of GRantagonist/agonist treatment on spinal cell activation after an acute inflammation Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison treatment vs control

14. mGluR5 Astrocytes 300 400 250 300 200 Density of mGluR5 (% left/right side) Density of GFAP (% left/right side) 150 200 100 100 50 0 0 L5 L6 L5 L6 level level Effect of GRantagonist/agonist treatment on spinal cell activation after an acute inflammation *** *** ** *** * *** *** Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ Data are shown as mean + SD. *P<5%, ***P<0.1%, t-test 2-tailed; comparison treatment vs control

15. *** 200 *** 180 *** 160 *** Δpaw volume (% control) 140 *** 120 *** *** *** 100 80 pre treat. pre inf. 1 4 7 10 14 *** time (day) *** *** *** 100 100 *** *** *** *** *** 80 *** 80 *** *** 60 *** *** Δforce (% control) Δpaw withdrawal latency (% control) *** *** 60 40 *** 40 von Frey test Plantar test *** 20 *** *** 20 0 pre treat. pre inf 1 4 7 10 14 pre treat. pre inf. 1 4 7 10 14 time (day) time (day) Effect of GRantagonist/agonist treatment on behavioural tests after a chronic inflammation Paw volume Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ *** *** Data are shown as mean + SD. ***P<0.1%, 1-way ANOVA followed by a Bonferroni test; comparison treatment vs control

16. 180 FosB/ΔFosB TNFα 140 160 *** *** *** *** 120 140 120 100 100 80 Number of TNFα-ir cells (left-right side) Number of FosB/ΔFosB-ir cells (left-right side) 80 60 60 40 40 20 20 *** *** *** *** 0 0 L5 L6 L5 L6 level level Effect of GRantagonist/agonist treatment on spinal cell activation after a chronic inflammation Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ Data are shown as mean + SD. ***P<0.1%, t-test 2-tailed; comparison treatment vs control

17. astrocytes mGluR5 400 300 250 300 200 Density of mGluR5 (% left/right side) Density of GFAP (% left/right side) 150 200 100 100 50 0 0 L5 L6 L5 L6 level level Effect of GRantagonist/agonist treatment on spinal cell activation after a chronic inflammation ** ** ** * * Dexamethasone (n=7): ■ RU486 (n=7): ■ Control (n=5): ■ Data are shown as mean + SD. * P<5%, **P<1%, t-test 2-tailed; comparison treatment vs control

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

19. Synopsis Sacrifice Pre-inflammation (d -2, -1, & 0) -2 0 1 7 -7 4 10 14 Time (d) Pre-stress (d -10, -9 & -8) • 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) Animal model: Wistar rats

20. 180 160 *** * *** 140 paw volume (% control) 120 100 Paw volume 80 Time (day) Pre stress Pre Inf. 1 4 7 10 14 *** *** 100 100 *** *** *** 90 90 80 80 *** *** 70 70 Force (% control) *** PWL (% control) 60 60 50 50 40 40 von frey test Plantar test *** *** 30 30 Time (day) Time (day) Pre stress Pre Inf. 1 4 7 10 14 Pre stress Pre Inf. 1 4 7 10 14 Effect of chronic restraint stress on behavioural tests after a chronic inflammation 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. TNFα FosB/ΔFosB 80 40 *** *** *** *** 60 30 Number of FosB/ΔFosB-ir cells (left-right side) Number of TNFα-ir cells (left-right side) 40 20 20 10 0 0 L5 L6 L5 L6 Level Level 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

22. microglia astrocytes 300 300 *** *** *** *** 250 250 200 200 Density of GFAP (% left/right side) Density of Iba1 (% left/right side) 150 150 100 100 50 50 0 0 L5 L6 L5 L6 Level Level Density of mGluR5 350 300 *** *** 250 200 Density of mGluR5 (% left/right side) 150 100 50 0 L5 L6 Level 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

23. Early phase of inflammation Pain inhibition (Gamaro et al., 1998 Machelska et al., 2003) ↑ opioid activity 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). ↓ opioid activity (Dantas et al., 2005) ↑ pain sensitivity Late phase of inflammation OR: Descending inhibitory pathway? (Watkins et al., 1982) Discussion Stress

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: • prospectivepreclinicalstudies • experimental psychophysicalstudies

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!!!