Stopping Pain in Its Tracts

Karin N. Westlund High, PhDUniversity of Kentucky

Disclosure Information

Karin Westlund High, PhD

Pfizer - Release of Patent RightsWorld Institute of Pain - Lecture

Honorarium

No discussion of unlabeled uses

Learning Objectives:

(1) Re-define Pain Pathways• Clinical Lesion Studies• Anatomical Tract Tracing• Electrophysiology• Imaging

(2) Treatment Strategies Utilizing Pain Pathways

• Arresting Neurogenic Inflammation• Gene Therapy

Relief from Joint and Visceral Pain

From Clinic to Laboratory

From Laboratory to ClinicFrom Laboratory to Clinic

Alleviation of pain can begin with re-definition of pain pathways

Multiple Pain Transmission Pathways

Ascending PathwaysDescending Pathways

Halting Dorsal Root ReflexesTarget Tissue Communication

Discriminitive InputNociceptive Input

Novel findings aboutVISCERAL PAIN:

• Neuronal Pathways• Neurophysiological Mechanisms• Behavioral Responses• Brain Activation Sites• Translational Strategies for Relief

of Visceral (Internal Organ) Pain

IntraoperativeView

Myelin SheathStain Showing

Area ofDemyelination

Drawing ofDemyelinatedArea in Dorsal

Column

Dorsal Column Lesion Relieves Cancer Pain

Guildenberg and Hirshberg, 1984 14 cancer patients

Hirshberg et al., 1996 8 cancer patients

Nauta et al., 2000 6 cancer patients

Becker et al., 1999 1 cancer patient

Kim and Kwon, 2000 5 thoracic cancer patients

Hwang et al., 2004 4 cancer patients

Total 38 patients

Midline MyelotomyMicroprobe Lesion of Dorsal Column Midline

Significant Reduction of

Pain VAS Scores 10 2.5-3

Narcotic Usage by 82%

Neurological Deficits14 cancer patients Guildenberg and Hirshberg, 1984 8 cancer patients Hirshberg et al., 1996 6 cancer patients Nauta et al., 2000

Cancer Pain

X

Postsynaptic Dorsal Column Cells

Hirshberg et al., Pain 67:291, 1996

DYE

Wang et al., J Comp Neurol, 2000

RetrogradelyRetrogradelyDYE

AntrogradelyAntrogradely

Wang, Willis, Westlund, J. Comp. Neurol. 415:341, 1999

Sugiera, 1990

Lateral Pain Pathways

Medial Pain PathwaysPSDC

LateralSTT

VentrolateralVentromedialSTT

Transmission of Afferent Input from

Skin

Muscle

Viscera

Vasculature

PAIN

PATHWAYS

Animal models of pancreatitisTranslational research is dependent on Translational research is dependent on developing a model which possesses a developing a model which possesses a longer time course in order to evaluate longer time course in order to evaluate central and peripheral sensitization central and peripheral sensitization processes.processes.

• Most animal models of pancreatitis are difficult to produce, short lived and/or too invasive

• Visceral pain is an activity-dependent condition dependent on inflammation, chemical insult or distension

Persistent Pancreatitis Models

DBTC DBTC Chemical Insult Chemical Insult

1 week1 week

High Fat Diet/High Fat Diet/EthanolEthanol

10 weeks

i.v.i.v.

Serum Lipase & AmylaseSerum Lipase & AmylasePancreas HistologyPancreas Histology

ImmunohistologyImmunohistology

ElectrophysiologyElectrophysiologyfMRI ImagingfMRI Imaging

PharmacologyPharmacology

Gene TherapyGene Therapy

HSVHSV--ENKNociceptiveNociceptivetestingtesting

ENK

Persistent Pancreatitis Activates Specific Regions Innervated

Periaqueductal GreyPeriaqueductal GreyRVMedullaRVMedulla Medial ThalamusMedial Thalamus Lateral ThalamusLateral Thalamus

Pancreatitis Activated Cortical Regions

fMRI Imaging of Visceral Pain

VISCERALVISCERALNOCICEPTIVE NOCICEPTIVE

PATHWAY NEURONS PATHWAY NEURONS BECOME SENSITIZEDBECOME SENSITIZED

CerveroCervero et al., 1992et al., 1992HirshbergHirshberg et al, 1996et al, 1996AlAl--Chaer et al., 1996Chaer et al., 1996Wang et al, 1999 Wang et al, 1999 Willis et al, 1999Willis et al, 1999Westlund, 2000Westlund, 2000AlAl--Chaer et al 1998 Chaer et al 1998 Houghton et al, 2001 Houghton et al, 2001 Wang & Westlund, 2001Wang & Westlund, 2001

pancreas

colon

Electrophysiology reveals that Electrophysiology reveals that prolonged noxious stimulation prolonged noxious stimulation sensitizes sites along the sensitizes sites along the pathwaypathway

Pain Transmission Pathways

Lateral Pain Pathways – cutaneous sources• Ventrolateral spinothalamic tract

Medial Pain Pathways – visceral sources• Dorsal column - midline and intermediate

septa• Ventromedial spinothalamic tract

200

150

100

50

0100

75

50

25

00 10 20 30 40 50 60

200

150

100

50

030

20

10

00 25 50 75 100 125 150 175 200 225 250

A

B

mm

Hg

mm

Hg

Spi

kes/

sec

Spi

kes/

sec

Time (secs)

Bradykinin

PIPRBR C

D

Bregma -14.30 mm

Gr

GrCu

Perc

enta

ge c

hang

e fr

om b

asel

ine

0

100

200

300

400

500

∗

n=11

Baseline Bradykinin

Noxious Pancreatic Stimulation Activates Dorsal Column Nuclei

Wang et al., 1999

Rat and Human Medial and Lateral

Spinothalamic Tracts &

Terminations

Visceral

Somatic & Visceral

V

VS

MehlerMehler, 1962; , 1962; Wang et al, 1999Wang et al, 1999; Ren and Westlund, submitted

Medial ThalamusCentral Lateral Thalamic Nucleus

Responds Only to Visceral Insults

Ren, Zhang and Westlund, submitted

10.0

7.5

5.0

2.5

0.00 25 50 75 100 125 150 175 200

Spi

kes/

sec

B Bradykinin

Dorsal column lesion

Time (secs)

10.0

7.5

5.0

2.5

0.00 25 50 75 100 125 150 175 200

Spi

kes/

sec

A Bradykinin Control

Time (secs)

Pancreatic Bradykinin Activates Lateral Thalamic Neurons Via the Dorsal Column

Houghton ,Wang and Westlund. Pain 89:207-220, 2001

0

100

200

300

400 Control DC lesions

Perc

enta

ge c

hang

e fr

om b

asel

ine

n=7

*

∗

Baseline

Effect of DC lesions on VPL cells to BK

VPM

VPL

Bregma –2.80 mm

Descending Reticulospinal

Pathways

APT Anterior Pretectal Nuclei

PAG Periaqueductal Gray

A5-A7 Pontine Noradrenergic Cells

SRD Subnucleus Reticularis Dorsalis

RVM Rostral Ventromedial Medulla

SRD

Ascending Only

Descending

Westlund and Coulter, Br Res Rev 2:235, 1980 Westlund et al., Prog Br Res 57:239, 1982 Westlund et al., Frontiers Clin Neurosci 4:55, 1984

PontineNoradrenergic Descending PathwaysRetrograde Transport of noradrenergic enzyme dopamine-β-hydroxylase

SPINOTHALAMIC TRACT CELLS

Westlund et al., J Comp Neurol 322:519-527, 1992

3-D Reconstruction 8 EM sections

STT dendrite

Axons

High Threshold

Wide Dynamic Range

Monkey Lumbar Spinal Cord

Catecholamine Synaptic Contacts 8%

Synaptic Contacts on

SpinothalamicTract Cells

Spinothalamic Tract from Lamina I

Contacts

• Noradrenergic• Serotonin• Glutamate 50%• GABA 30%• CGRP

Noradrenergic Cell Groups A1-A7

Westlund et al., J Comp Neurol, 299:178 1990 Westlund et al., J Comp Neurol, 322:519, 1992 Carlton et al., J. Comp Neurol, 322:528, 1992 Carlton et al., Neurosci Letters, 109:76, 1990

Westlund and Craig, Exp Brain Res110:151, 1996

Central and Peripheral Mechanisms of Inflammatory

Joint Pain

A Missing Link in Treatment is

Consideration of Neurogenic and Neuroimmune

Activation in Arthritis

NEUROGENIC MEDIATORS of INFLAMMATION

•• Substance P (SP)Substance P (SP)-- Plasma extravasationPlasma extravasation-- VasodilationVasodilation-- ChemoattractantChemoattractant for macrophagesfor macrophagesCalcitoninCalcitonin genegene--related peptide (CGRP)related peptide (CGRP)-- VasodilationVasodilationGlutamate (Glutamate (GluGlu) and Aspartate (Asp)) and Aspartate (Asp)-- VasodilationVasodilation-- Increased nociceptionIncreased nociception-- Increased Increased bloodflowbloodflow

•• Other Neurotransmitter ReceptorsOther Neurotransmitter Receptors-- TRPVR1, TRPVR4, TRPA1 TRPVR1, TRPVR4, TRPA1 -- Acid sensing GAcid sensing G--protein coupled receptorprotein coupled receptor

CGRPSP

EAA H+

TRP 2

1 non1 non--NMDANMDA2 GABA2 GABAAA3 NMDA3 NMDA

ThalamusThalamusBrainstemBrainstem

DRG

3

1Dorsal Root Dorsal Root

ReflexesReflexes

Neurogenic Amplification Neurogenic Amplification of Painof Pain

NeuroNeuro--immune immune Factors Factors in Painin Pain

Spinal Sensitization

Vicious Cycles Driving Amplification of Pain

Dorsal Root Reflexes• Half of inflammation is initiated centrally by

and does not occur in denervated tissue• Can be blunted centrally

Neurogenic Inflammation• Neurotransmitter receptors on non-neural

tissues initiate release of inflammatory mediators

• Inflammation increases neurotransmitter receptors for feedforward effect

Dorsal Root ReflexesDRRs are induced by

• K+ Channel Activator AP4• Joint Flexion• Brisk mechanical cutaneous stimulation• Arthritis models

Acute K/C model ipsilaterallyChronic CFA bilaterally

DRRs are Inhibited by• Lidocaine• Dorsal Rhizotomy• Spinal CNQX, AIDA, gabapentin or bicuculline

are not inhibited by GABA B or NMDA antagonists

DRRs are recorded from all fiber typesDRRs require continues peripheral input

DORSAL ROOT AXONAL REFLEXES (DRR)

GLU

GLU Sensitization

Sluka, Willis and Westlund, JPET 271:76, 1994; Sluka, Willis and Westlund, Pain Forum 4:141, 1995

NEUROGENIC INFLAMMATION

Dorsal Root Reflexes in

Medial ArticularNerve

in Arthritis Models

Rees, Sluka,Westlund and

Willis, 1994

Gabapentin, GABAA, non-NMDA, mGlu Blockers Diminish Dorsal Root Reflexes in Arthritic Rats

Kaolin/carrageenan-induced arthritis can produce DRR spontaneous activity

Sluka et al., 1994; Rees et al., 1995a,b Willis, Sluka, Rees and Westlund, 1998 Zhang et al., 2000 Lawand, Willis and Westlund, unpublished

K / C INDUCED ARTHRITIS

Arthritis Glutamate antagonist

Sympa-thectomy

Dorsalrhizotomy

Experimental Arthritis

Reduced inflammation

Central

Sluka and Westlund, 1993; Sluka et al., 1994

Neurotransmitter Release Parallels Inflammation Induced Sensitization

Occurs Centrally and PeripherallyP

ER

CE

NT

CH

AN

GE

Injury

Secondary Hyperalgesia

Neurotransmitter Release

TIME

1. Minutes Formalin/capsaicin/BK

2. Hours Kaolin/carrageenan

3. Days Complete Freund’s adjuvant

↓GLU

X

Lawand, McNearney, Westlund, Pain 86: 69, 2000Westlund et al., 1992

SYNOVIAL FLUID GLUTAMATE

TIME (hrs)1 2 3 4

Perc

ent c

hang

e of

gl

utam

ate

from

bas

elin

e

0

50

100

150

200

250

300

350

400

K/C-treated (no lidocaine)K/C-treated +lidocaineSaline-treated

Base

k/c

∗∗

∗

∗

∗

∗

0

10

20

30

40

50

60

Glu Gln Gly Ctn

SF A

A c

once

ntra

tions

Pa

tient

/Cad

aver

s

*

Mean SF Amino Acid Concentration (mM)

Synovial Fluid from Arthritic Patients

*

Asp Ser Arg Thr

McNearney et al., J. Rheumatology 27: 739, 2000

Animal Models of Neurogenic Arthritis

GLU

n=2

Time (min)5 10 15 20 25 30 35 40 45 50

% C

hang

e fr

om b

asel

ine

-200

0

200

400

600

800 Phosphate Buffer Glutamate 1mM

GlutamateGlutamate--Induced Blood FlowInduced Blood Flow

GLUGLUGLUGLU

Intraarticular injections of combinations of amino acids:

Effects on reflexive pain assessmentsPBASP/GLU ASP/ARG ASP/GLU/ARG

TIME (HOUR)inj

LATE

NC

Y (S

EC)

6

7

8

9

10

*

*

*

* **

*

*

*

**

base

*

**

1 2 3 4 5 6 7 8

*

***

TIME (HOUR)

WIT

HD

RA

WA

L TH

RES

HO

LD (m

N)

inj

20

40

60

80

100

base

**

*

***

*

*

*

*

*

*

**

*

1 2 3 4 5 6 7 8

*

Secondary Mechanical Hyperalgesia and AllodyniaLawand et al. , Eur J Pharmacol, 324:169, 1997

CONCLUSIONS

•• Neurogenic inflammation is partially Neurogenic inflammation is partially the result of direct release of the result of direct release of glutamate in the spinal cordglutamate in the spinal cord

•• Neurogenic inflammation is amplified Neurogenic inflammation is amplified by the effects of axon reflexes and by the effects of axon reflexes and peripheral glutamate releaseperipheral glutamate release

HUMAN ARTHRITIS In Vitro MODEL

Human Clonal Synoviocytes SW982 Have Neurotransmitter Receptors

and

Respond with Release of Inflammatory Mediators

Control 24h NMDA 5µM

Human Clonal SynoviocytesAn in vitro Arthritis Model

NMDA NR1 Receptor

Glutamate Reactivates Inflammatory Proteins in Human

Primary Synovial Cells

McNearney et al., Clin Exp Immunol. 137: 621-627, 2004

SF (-) (+) SF (-) (+)0

10

20

30

40

50

60

*

RAGout

^

(+/-) Added L-Glutamate

Mea

n TN

F-α

Lev

els,

pg/

ml

NMDA NR1 is Upregulated in Rat Spinal Cord after CFA-induced Arthritis

020406080

100120140160180200

Normal Arthritis

Nor

mal

ized

Inte

grat

ed

Dens

ity

NR11 2 3 4 1 2 3 4

DAY 7

Glutamate Receptor NMDA NR1

213 Kd

134 Kd

82 Kd

40 Kd

1 2 3 4 5

– + – +Brain Synoviocytes

NMDA+ACPD

NR1, Synoviocyte

NR1, Brain

Vimentin

β-Actin

STD

Std Brain Synoviocytes

- +

β-Actin

NR1, Synoviocytes

Increased after NMDA+ACPD

NR1, Brain

L-Glutamate Upregulates TNF-α levels in CulturedPrimary Synovial Fluid Cultures From Patients

GoutGout Rheumatoid ArthritisRheumatoid Arthritis

0

5

10

15

20

25

30

35

40

Bkgr - Glu + Glu -Glu + Glu

SEMAvg

0

50

100

150

200

250

Bkgr - Glu + Glu -Glu + Glu

SEMAvg

1 2 1 2

1. Initial 2 Hr Treatment

2. Re-Activation after 3 weeksMcNearney and Westlund, submittedMean Supernatant TNF-α Levels, in pg/ml

TNF Potentiation

TNF-α potentiates calcium response to prolonged noxious (45 oC) heat

A: control (vehicle treated) synoviocyte

B: TNF-α (1 ng/ml, 8 hr) pretreated cells

C: Peak and average calcium response in control (white bars, n=7), or TNF-α(1 ng/ml) treated synoviocytes.

+TNF-α

Inflammatory Mediator Increases TRP Receptors

Synoviocytes Have TRP Receptors

Human Synoviocytes are Responsive to pH HypoOsmolarityThermal Stimulation (Hot & Cold)

H+

H+H+

H+

H+EDEMA HEAT COLDACID

RT-PCR Detection of TRP channel mRNAHuman

*Human clonal & primary synoviocyte cultures were done in parallelKochukov et al., 2007

Clonal

Primary

TRPV1 Receptors

Human Synoviocytes Have Proton Sensing G Protein-Coupled Receptors

1.4

1.2

1.0

0.8

0.6

Rat

io (3

40 n

m/3

80 n

m)

2000s150010005000 sec

pH 6.4 pH 6.4 pH 6.4 pH 5.5 pH 5.5

Intact cells were incubated in pH 7.4 standard bath saline. Acid pH solutions were applied from the pipette placed ~ 400 µ M from a cell.

Christensen, Kochukov, McNearney, Taglialatela, Westlund, 2005

GLU

TRP ⇒ COX-2

GLU ⇒ TNFα ⇒ TRPV1

Injury / Oxidative Stress/ ↓pH ROS / NO/ Edema

VICIOUS CYCLES OF NEUROGENIC INFLAMMATION

Sensitization

Peripheral Sensitization

GLU

CGRPCGRPSPSP

EAA EAA ΗΗ++

TRPTRP2

1 non1 non--NMDANMDA2 GABA2 GABAAA3 NMDA3 NMDA

ThalamusThalamusBrainstemBrainstem

DRG

3

1DRRsDRRs

Are we treating all the PainAre we treating all the Pain

Sluka, Willis and Westlund, JPET 271:76, 1994; Sluka, Willis and Westlund, Pain Forum 4:141, 1995

GENE THERAPY APPROACHES

for TREATMENT of VISCERAL PAINHerpes Simplex-1 (HSV)Injections Targeting

Knee JointPancreas

Transport to dorsal root ganglia for overexpressionof met-Enkephalinprovides reduced

Behavioral responsesInflammationTissue Damage

Gene Therapy to Relieve Arthritis Pain

Opiate-like Peptide Delivery

Intensity

0 50 100 150 200 250

Pain

Sen

sitiv

ity0

20

40

60

80

100

120

Normal Arthritis + Gene Therapy

Arthritis

Intensity

0 50 100 150 200 250

Pain

Sen

sitiv

ity0

20

40

60

80

100

120

Normal Arthritis + Gene Therapy

Arthritis

Relieves Arthritis Pain

DRG

Lu, McNearney, Wilson, Yeomans, Westlund, Eur J Neurosci, 27:1153, 2008

Met-EnkephalinSpinal Cord 1 week after induction

of DBTC pancreatitis

DBTC/HSV-ß-gal DBTC/HSV-ENK

Cord (T10)

0

1

2

3

4

5

6

Enke

phal

in (n

g/m

l)

veh ic le /hsv-enk (n= 1)db tc /hsv-b -ga l (n= 1)db tc /hsv-enk (n= 1)

Lu et al. Mol Therapy, 2007

1 week after induction of DBTC-induced pancreatitis

Sham Surgery DBTC/vehicle

DBTC/met-ENKDBTC/β-gal

Pancreas Expression of

Lu et al. Mol Therapy, 2007

HSVHSV--ENKENKRestores Restores Open Field Open Field Exploratory Exploratory Behavior in Behavior in Acute Acute Pancreatitis Pancreatitis ModelModel

Lu et al. Mol Therapy, 2007

HSV

ENK

C E β E β E β0

100

200

300

400

500ControlHSV-EnkHSV-β -gal

400 40 4 pfu/ml

**

***

24 Hour Incubations

Mea

n TN

F-α

Con

cent

ratio

n, p

g/m

l

Pancreas

Yang et al. Mol Pain, 2008Lu et al. Eur J

Neuro, 2008

Tissue Protection

Reduced Inflammation

SignificanceUnique advantages offered by replication

defective HSV-1 viral vectors for transgeneexpression of met-enkephalin by peripheral

nerves?

Vector remains outside the CNS in the DRG

Effective reduction of pain measures

Prolonged effectiveness of 4-6 weeks or longer

Reduction/elimination of the use of narcotic drugs

Focused delivery to the target organ of low viral titerslimits side effects typical of opiate therapies

Basic ScientistsElie Al-ChaerWilliam Willis Ying Lu Alan WangAndrea HoughtonLiping ZhangNada Lawand

Visceral Models and Pathways

Clinical ScientistsRobert HirshbergHank NautaElena HewittCourtney WilliamsGerry CampbellVicki SoupkupMatt SmileyAmr ZidanJames LinRod Fabian

Neurogenic InflammationPrimate Central Sensitization

Kathleen Sluka

Pat Dougherty

Linda Sorkin

Sue Carlton

Arthritis Model and DRRs

Kathleen Sluka

Ying Lu

Huw Rees

William Willis

Jing Wu

Liping Zhang

Medial Articular Nerve

Nada Lawand

William Willis

In vitro Arthritis Model

Terry McNearney

Mikhail Kochukov

Burgess Christensen

Human StudiesTerry McNearney

HSV Gene Therapy Collaborative Team

University of Texas University of South CarolinaMedical Branch Steven P. Wilson, Ph.D.

Rong Chu, B.A.Weidong Lin, B.A. Yong Ren, Ph.D. Stanford UniversityHong Yang, M.D. David Yeoman, Ph.D.Ying Lu, M.D. Terry McNearney, M.D.

Citius, Altius, Fortius, Purius