Mechanisms of visceral pain and Mechanisms of visceral pain and hyperalgesiahyperalgesiaMechanisms of visceral pain and Mechanisms of visceral pain and hyperalgesiahyperalgesia

Fernando CerveroFernando Cervero

Director, The Alan Edwards Centre for Research on PainDirector, The Alan Edwards Centre for Research on Pain

McGill University, McGill University, Montreal, CanadaMontreal, Canada

President, IASPPresident, IASP

Working Working together together for pain relief for pain relief throughout the worldthroughout the world

Visceral pain is the most frequent form of pain

Few (if any) analgesics specifically directed at visceral pain conditions

• Sociological reasons:

VISCERAL PAIN and ANALGESIA

� Patients often seen by practitioners with no primary interest in pain

treatment

• Biological reasons:

� inadequate animal models of visceral pain

� unawareness of specifics of visceral pain

Sleep and pain tend to inspire poets and philosophers

(micturition and defecation do not)

An unglamorous kind of pain

with psychoanalysts is the other way round

(Peter Nathan)

Mechanistic features of visceral Pain

• Sensory receptors / Nociceptors:

– Intensity encoders as well as high threshold (summation?)

– Chemosensitive ( ischemia sensitive)

– Sensitization + profound changes in microenvironment (secretory, motility, hormonal…)

– Many “non-sensory” receptors (CNS actions). Can they become sensory under pathological conditions ? (IBS?)

SP in a mucosal afferent ending (ureter)

Semenenko & Cervero (1992)

Thresholds of ureteric afferents

Cervero & Sann (1989)

Fullness

Pain

Bladder reflexes

LUMEN BLADDER

TRPV1

vanilloids

Urothelial

Role of the urothelium in the signalling of sensory events in the bladder

Bladder reflexes

ATPP2X3

distension

TRPV1

?

?Primary afferent neuron

Urothelial cell

Effect of the presence of an experimental calculus on the motility of the rat ureter in vivo.

(Laird, Roza & Cervero 1997)

• No role in responses to acute mechanical stimuli

• No role in responses to non-neurogenic inflammatory stimuli

• Required for sustained behavioral responses to neurogenic inflammatory stimuli

Role of Nav1.8 in hyperalgesia

neurogenic inflammatory stimuli

Therefore:

• Nav1.8 contributes to spontaneous activity in sensitized nociceptors

Laird, et al. (2002) J.Neurosci. 22, 8352

CoCo--expression of CBexpression of CB11 and P2Xand P2X33 receptorsreceptors

• CB1 receptors are expressed in the urothelium and in nerve fibers of the

muscular and sub-urothelial layers• CB1 and P2X3 are co-expressed in the urothelium

CB1 receptor P2X3 receptor Merged

L L LL L L

L L L

L L L

L = lumenBar = 20µm

Negative c

ontr

ol

CBCB11 receptorreceptor TRPVTRPV11 receptorreceptor MergedMergedO

vera

ll

L L L

CoCo--expression of CBexpression of CB11 and TRPVand TRPV11 receptorsreceptors• CB1 and TRPV1 are co-expressed in umbrella cells• Some nerve fibers of the sub-urothelial and muscular layers co-express CB1

and TRPV1 receptors

L L L

Musc

ula

r la

yer

Uro

theliu

m +

Suburo

thelia

l

layer

*

*

*

*L = lumenBar = 20µm

CBCB11 receptorreceptor Substance PSubstance P MergedMergedO

vera

ll

L L L

CoCo--expression of CBexpression of CB11 and Substance Pand Substance P• CB1 and Substance P are co-expressed in nerve fibers of the sub-urothelial layer.• Sparse co-localization of CB1 and Substance P in the muscular layer.

L L L

Musc

ula

r la

yer

Uro

theliu

m +

Suburo

thelia

l

layer

L = lumenBar = 20µm

Number of abdominal contractions

(during 5 minute periods)

Number of micturitions

(during 20 minute periods)

12

14

16

18

20

Saline

CYP

8

10

12

14Saline

CYP

Cyclophosphamide Cystitis: BehaviorCyclophosphamide Cystitis: Behavior

0

2

4

6

8

10

12

0

2

4

6

8

**

Firing threshold (mm Hg)

120

140

160

180

Volume infused

to reach 40 mm Hg (µl)

8

10

12

Cyclophosphamide Cystitis: PhysiologyCyclophosphamide Cystitis: Physiology

**

*

0

20

40

60

80

100

120

Normal CYP

0

2

4

6

8

Normal CYP

Normal CYP pre AZ12646915 CYP post AZ12646915

0

300

40Intravesical

pressure (mm Hg)

Afferent

activity

(spikes/s)

10

0 µ

V

10 s

Waveform

Cannabinoid agonist (CBCannabinoid agonist (CB11/CB/CB22) reverses sensitization) reverses sensitization

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

Normal

CYP pre AZ12646915

CYP post AZ12646915

Intravesical pressure (mm Hg)

Aff

ere

nt

act

ivit

y (

spik

es/

s)

+

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40

CYP pre AZ12646915

CYP post AZ12646915

Intravesical pressure (mm Hg)

No

rma

lize

d a

ctiv

ity (

%)

(ve

rsu

s p

re-d

rug

ma

xim

al

act

ivit

y)

Sensitization reversal is mediated by CBSensitization reversal is mediated by CB11 receptorsreceptors

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40

CYP pre

AZ12646915 +

AM630CYP post

AZ12646915 +

AM630

Intravesical pressure (mm Hg)

No

rma

lize

d a

ctiv

ity (

%)

(ve

rsu

s p

re-d

rug

ma

xim

al

act

ivit

y)

0

20

40

60

80

100

120

140

0 5 10 15 20 25 30 35 40

CYP pre

AZ12646915 +

AM251

CYP post

AZ12646915 +

AM251

•• Cannabinoid receptor CBCannabinoid receptor CB11 mRNA is present in the urinary bladder mRNA is present in the urinary bladder

(urothelium, and nerve fibers). CB1, TRPV1 and P2X3 (urothelium, and nerve fibers). CB1, TRPV1 and P2X3 receptors are receptors are coco--expressed.expressed.

•• Intravesical administration of a cannabinoid receptor agonist reduces the Intravesical administration of a cannabinoid receptor agonist reduces the mechanicallymechanically--evoked activity of bladder afferents. This effect is evoked activity of bladder afferents. This effect is abolished by the previous administration of a CB1 antagonist.abolished by the previous administration of a CB1 antagonist.

• Afferent activity in inflamed bladders is increased for intravesical pressures

Cannabinoids and visceral nociceptorsCannabinoids and visceral nociceptors

• Afferent activity in inflamed bladders is increased for intravesical pressures between 10 and 40 mmHg. Local treatment with a CB1/CB2

cannabinoid agonist significantly reduces afferent activity at intravesical pressures above 20 mmHg. This effect is mediated by CB1

receptors.

•• Cannabinoid CBCannabinoid CB11 receptors are implicated in the peripheral modulation of receptors are implicated in the peripheral modulation of bladder sensory information by a bladder sensory information by a direct action of cannabinoids on nociceptive afferents

Mechanistic features of visceral Pain

• CNS / central organization:

– Evidence against brief/short lived nociceptive pain (no visceral “tail flick” equivalent)

– Alarm system with widespread motor and autonomic reactions (“trip-wire” arrangement)

– fMRI data: some (relatively minor) differences in activated brain areas (mostly concerned with non-sensory aspects: emotional, affective)

Viscero-somatic convergence in the spinal cord

Pain and spinal cord Pain and spinal cord hyperexcitabilityhyperexcitability

““the irritable focusthe irritable focus””

From: J. MacKenzie (1909)

…..this portion of the spinal …..this portion of the spinal cord may be looked upon as cord may be looked upon as cord may be looked upon as cord may be looked upon as being rendered abnormally being rendered abnormally excitable in consequence of a excitable in consequence of a violent stimulation from the violent stimulation from the organ V…..organ V…..

CNS

How visceral pain worksHow visceral pain works

P

Aδδδδ / CPrimaryPrimaryHyperalgesiaHyperalgesia

Aββββ

Nociceptor

sensitization

1

Synaptic

strengthening by

incoming

afferent volleys

(sensitization)2

AllodyniaReferredReferredHyperalgesiaHyperalgesia Aδδδδ / C

T

Aββββ

P

Aδδδδ / CActivation of

nociceptive

neurons by LT

afferents3

Synaptic strengthening by incoming afferent volleys (sensitization)

Visceral Hyperalgesia and CNS hypersensitivity:

Role of glutamate receptor trafficking

A. Contractor, S.F.Heinemann Sci. STKE 2002, RE14 (2002)

Visceral painful stimuli recruit GluR1 AMPA receptor subunits to the membrane

GluR1

B 10 45 90 180

GluR2/3

MEMBRANE

2

3

4

5

*** **

** GluR1

CYTOSOL

GluR1

B 10 45 90 180

GluR2/3

Capsaicin (min)

B 10 45 90 180

Capsaicin (min)

Galan, Laird and Cervero, 2004

10 45 90 1800

1

Time after capsaicin instillation (min)

GluR2/3

Mobilization by exocytosis?

Inhibition of the exocytosis pathway blocks recruitment of

GluR1 to the membrane and reduces referred hyperalgesia

MEMBRANE

B Vh BFA

Brefeldin A given i.t. 10 min before intracolonic capsaicin

45 min

80

100 Baseline

Vh + CapsaicinCaps + 15 nmoles BFA

Capsaicin

Galan et al , 2004

1 4 8 16 320

20

40

60

****

****

Force (mN)

0.0

0.5

1.0

1.5

2.0

Capsaicin

**

#

AMPA trafficking and visceral painAMPA trafficking and visceral pain

• Acute painful stimuli induce trafficking (membrane delivery) of GluR1 AMPA receptors in vivoGluR1 AMPA receptors in vivo

• Inhibition of AMPA trafficking reduces secondary hyperalgesia induced by acute stimuli

Functional Pain

Spontaneous and persistent pain in the

absence of an apparent cause

• Frequent form of chronic pain (Irritable Bowel Syndrome, Interstitial Cystitis, (Irritable Bowel Syndrome, Interstitial Cystitis, Chronic Pelvic Pain, Fibromyalgia….)

>30% all chronic pain

• More prevalent in women (> 65%)

• Unknown mechanisms (a role for estrogen?)

Functional Pain and HyperalgesiaFunctional Pain and Hyperalgesia

Spinal Cord

Aββββ

Genetic factorsPersistent painHormonesCognitive factors

SecondarySecondaryHyperalgesiaHyperalgesia

AllodyniaAδδδδ / C

T

Aββββ

P

50

75

100

OVX

Control

Sham

req

uen

cy (%

)

50

75

100

* ** *

req

uen

cy (%

)

OVX induces a long lasting abdominal mechanical hyperalgesic state

1 4 8 16 320

25

50

mN

Res

po

nse

fr

1 4 8 16 320

25

50

*

mNR

esp

on

se fr

Week 1 Week 5

OVx – Preventive Hormone Replacement Therapy

25

50

75

100R

esp

on

se f

req

uen

cy (%

)

25

50

75

100

***

**

Res

po

nse

fre

qu

ency

(%)

OVX + 17β-Estradiol

OVX + placebo

Control

Sham

1 4 8 16 320

mN

R

1 4 8 16 320

mN

R

Week 1 Week 5

Prevention of abdominal mechanical hyperalgesia(pellets on week 1)

OVx – Restorative Hormone Replacement Therapy

25

50

75

100

Res

po

nse

freq

uen

cy (%

)

25

50

75

100

Res

pon

se fr

equ

ency

(%)

25

50

75

100

Res

po

nse

freq

uen

cy (%

)

OVX + 17β-Estradiol

OVX + placebo

Control

Sham

Week 1 Week 5 Week 6

Reversal of abdominal mechanical hyperalgesia(pellets on week 5)

1 4 8 16 320

mN

1 4 8 16 320

mN1 4 8 16 32

0

mN

(Sanoja and Cervero, 2008)

Activation of ERK1/2 in OVX mice (7 weeks post-surgery)

Lumbo-Sacral Spinal Cord

C S OVX

C S OVX

- p-ERK 1/2 (44/42 kDa)

- ERK 1/2 (44/42 kDa) -

GAPDH (37 kDa) -

C S OVX

*

Activation of ERK1/2 in OVX mice (estrogen reversal)Lumbo-Sacral Spinal Cord

p-ERK 1/2 (44/42 kDa)

ERK 1/2 (44/42 kDa)ERK 1/2 (44/42 kDa)

*

Visceral Pain

– The most frequent form of clinically relevant pain

– Inadequately managed and poorly treated

– Mechanistically: an alarm system with widespread motor and autonomic reactions

– Periphery: close relation with visceral function (motility, secretion, epithelial transport)

– CNS: hormonal / metabolic influences – slow time course

– Animal models of visceral pain should reproduce a functional process

Whereas acute symptomatic pain serve

the useful purpose of warning, chronic

pain is a malefic force which imposes

severe emotional, physical and economic

stresses on the patient

John J. Bonica

John J. John J. BonicaBonica (1917(1917--1994) 1994)

What to do to relieve pain?

- Increase our knowledge: Study pain

- Apply our knowledge: Treat pain

- Create a communication vehicle

between those who study and

John J. John J. BonicaBonica (1917(1917--1994) 1994)

between those who study and

those who treat pain

Working Working together together for pain relief for pain relief throughout the worldthroughout the world

www.IASP-pain.org