Endogenous opioid-mediated antinociception in cholestatic mice is peripherally, not centrally,...

Endogenous opioid-mediated antinociception in cholestatic

mice is peripherally, not centrally, mediated

Lisa Nelson1, Nathalie Vergnolle2, Charlotte D’Mello1, Kevin Chapman2,

Tai Le1, Mark G. Swain1,*

1Liver Unit, Gastrointestinal Research Group, Health Sciences Center, University of Calgary, 3330 Hospital Dr., NW, Calgary, AB, Canada T2N 4N12Mucosal Inflammation Research Group, University of Calgary, Calgary, AB, Canada T2N 4N1

Background/Aims: Cholestasis is associated with naloxone reversible antinociception and opiate receptor antagonists

are used clinically to treat pruritus. Pain and pruritus are closely interrelated and opioids modulate both sensations.

Therefore, we undertook a series of experiments to characterize opioid-mediated antinociception in cholestasis and

determine if it occurs inside or outside the CNS.

Methods: Antinociception scores to both thermal and mechanical stimuli were determined in mice with cholestasis

due to bile duct resection vs sham controls.

Results: Cholestatic mice demonstrated significant antinociception to both stimuli compared to controls, which

was reversible by the opiate receptor antagonist naloxone. The experiments were repeated with a naloxone

derivative, which does not cross the blood–brain-barrier (i.e. naloxone methiodide) with similar results, indicating

an opioid antinociceptive effect mediated outside of the CNS. Experiments with intraplantar injections of low dose

naloxone methiodide confirmed that cholestasis-associated antinociception occurs at the level of cutaneous nerve

endings. These findings were supported by findings of increased dermal met-enkephalin expression in cholestatic

mice.

Conclusions: Cholestasis in mice is associated with antinociception due to local effects of endogenous opioids (i.e. met-

enkephalin) at the level of sensory nerve endings. These findings may have direct implications in the management of

cholestasis associated pruritus.

q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Opioids; Met-enkephalin; Analgesia; Opioid receptor; Naloxone; C-fibers

1. Introduction

Pruritus is commonly encountered in patients with

cholestatic liver diseases and can be a profoundly bother-

some and disabling symptom [1,2]. However, the patho-

genesis of pruritus in cholestasis remains poorly understood.

Traditionally, cholestasis associated itch has been postu-

lated as resulting from the interaction of free unmyelinated

cutaneous nerve endings with a substance(s) retained in the

circulation as part of the cholestatic syndrome. The likely

0168-8278/$32.00 q 2005 European Association for the Study of the Liver. Pub

doi:10.1016/j.jhep.2005.11.043

Received 22 June 2005; received in revised form 17 October 2005;

accepted 7 November 2005; available online 27 December 2005* Corresponding author. Tel.: C1 403 220 8457; fax: C1 403 270 0995.

E-mail address: [email protected] (M.G. Swain).

unmyelinated neuron involved in this process is the recently

defined C-fiber, which carries itch related nerve impulses

from the skin to the spinal dorsal horn [3,4]. Afferent spinal

projections then carry the itch signals in the contralateral

spinothalamic tract to the thalamus and ultimately to the

somatosensory cortex [5]. These central neural connections

involved in itch allow for the modulation of pruritus and

potentially for the de novo induction of pruritus within the

CNS [5,6].

Opioids have well documented roles in both antinocicep-

tion and pruritus [7]. In addition, although pruritus and pain

are distinct sensations, there exist complex interactions

between them. Specifically, itch can be inhibited by pain

and inhibition of the central processing of pain can induce

itch [8]. Therefore, increasing severity of pruritus appears to

Journal of Hepatology 44 (2006) 1141–1149

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L. Nelson et al. / Journal of Hepatology 44 (2006) 1141–11491142

depend upon a fine balance between increased activity of

itch pathways and decreased activity in pain pathways [8,9].

Spinal neurons, which process pruritic signals are not

spontaneously active [4], whereas pain processing spinal

neurons are spontaneously active [8]. If spinal pain

processing neurons suppress itch processing neurons, then

inhibition of spinal pain neurons by opiates would be

expected to generate central itch [8,9].

In cholestasis there is clinical and experimental evidence

for both enhanced peripheral (i.e. outside the CNS) and

central opioidergic tone. Specifically, in cholestatic patients

and rodents there are increased levels of circulating

endogenous opioids [10,11]. In addition, there is evidence

of decreased opioid receptor numbers in brains of

cholestatic rats [12], and clinically, patients with cholestatic

itching can experience an opioid withdrawal-like syndrome

when given opioid receptor antagonists [13]. Moreover,

opioid receptor antagonists can ameliorate pruritus in

cholestatic patients [13,14]. Furthermore, cholestatic

rodents exhibit antinociception in response to a thermal

stimulus, which is reversed by the opioid receptor

antagonist naloxone [15]. No animal studies have reported

yet cholestasis-induced differences in nociceptive response

to mechanical stimuli.

Therefore, pruritus in cholestasis may be due to a

combination of the stimulation of cutaneous itch fibers by an

undefined pruritogen (possibly bile salts or histamine, levels

of both of, which are elevated in cholestasis [16,17], and the

action of endogenous opioids acting peripherally and/or

centrally to inhibit pain pathways. In support of this

hypothesis, opioid receptors have been identified on

cutaneous sensory nerve endings [18,19], and in patients,

opiate-mediated itch can be ameliorated by a opioid

receptor blocker, which does not penetrate the CNS [20].

Therefore, we undertook a series of experiments to, firstly,

further characterize cholestasis-associated antinociception

by examining different nociceptive stimuli (both thermal and

mechanical) and secondly, to determine whether cholestasis-

induced antinociception occurs as a result of opioid receptor

stimulation by opioids at peripheral (i.e. outside the CNS) or

central sites. To do this, we assessed anticociception in

cholestastic mice treated with opioid receptor blockers that

either act centrally and peripherally, or are excluded from the

CNS. Given the close association between pruritus and pain,

and the lack of suitable models with, which to study pruritus

directly in animal models of cholestasis, we used cholestasis

associated antinociception as a way to further understand

pruritus in cholestasis.

2. Materials and methods

2.1. Animals

Male C57BL/6 mice (6–8 week old) were obtained from Charles RiverLaboratories (Montreal, QC, Canada) and were housed in transparent

plastic cages in a room with a 12 h light/12 h dark cycle. Mice were fedstandard laboratory chow and tap water ad libitum. All experiments wereapproved by the Animal Care Committee of the University of Calgary andwere performed in accordance with the guidelines of the Canadian Councilon Animal Care.

2.2. Cholestasis induction

Under halothane anesthesia mice were either bile duct (cholestatic) orsham resected (control) as described previously [11,21]. Cholestasis wasconfirmed biochemically by determination of serum bilirubin and alanineaminotransferase levels using kits from Sigma.

2.3. Measurement of nociception

Nociceptive responses to thermal stimulation were evaluated bymeasuring paw withdrawal latency in response to a radiant heat stimulusapplied by a plantar test apparatus (Ugo Basile, Milan, Italy). Nociceptiveresponses to mechanical stimulation were assessed by examining theresponses to plantar stimulation with von Frey monofilaments with bendingforces of 0.166, 1.202 and 1.417 mN as previously described [22]. Briefly, ascore of 0 was given for no response, a score of 1 for withdrawal response,and a score of 2 for withdrawal response with licking or holding of the paw.All measurements were performed first under basal conditions (beforesurgery), and then at different time-points after surgery. Thermal analgesiawas defined as increased withdrawal latency compared to basal values.Mechanical analgesia was defined as decreased nociceptive score comparedto basal values.

2.4. Drug treatments

Systemic naloxone and naloxone methiodide (Sigma, St Louis, USA)treatments were given by daily sub-cutaneaous injections (dorso-scapularregion), at the dose of 1 mg/kg [15], the first injection startingimmediately after bile duct resection or sham surgery. Intraplantarinjection of naloxone methiodide was performed in a total volume of10 ml and at a dose of 15 mg/mouse [23]. The same volume of saline wasinjected into the paw of control mice. Intraplantar injections wereperformed 7-days after bile duct resection or sham surgery, andnociceptive responses were measured 30-min and 2-h after intraplantartreatments.

2.5. Paw Methionine(met)-enkephalin mRNA levels

and Immunohistochemistry

To determine whether the local production (i.e. intraplantar) ofendogenous opioids was increased in cholestatic mice, we removed pawtissues from BDR and sham mice 10 days post-surgery for determination ofpre-proenkephalin mRNA and met-enkephalin protein levels. We chose tospecifically examine met-enkephalin as previous reports have implicatedthis endogenous opioid in cholestasis associated itch [10]. Paw tissue waseither snap frozen on dry ice (for RNA extraction) or was placed in 10%formalin (for immunohistochemistry).

(i) Paw pre-proenkephalin mRNA expression: RNA was extracted fromindividual paw tissues removed from BDR and sham mice using TrizolReagent (Invitrogen, Burlington, ON) and steady-state pre-proenkephalinmRNA levels determined by RT-PCR using the ‘primer dropping’ method[24]. Pre-proenkephalin mRNA primer sequences were obtained fromMouse Genome Informatics (The Jackson Laboratories; MGI AccesssionNumber 1204569) which gave a product size of 141 bp. The internalhousekeeping gene control used was b actin with primers designed to give aproduct of 296 bp [25]. RT-PCR was performed using a ThermolyneAmplitron II cycler (Barnstead International, Dubuque, IO) with denaturing(30 s at 94 8C), annealing (30 s at 55 8) and extension (60 s at 72 8) with33 cycles for pre-proenkephalin and 24 cycles for b actin. RT-PCR productswere separated using a 2% agarose gel (Sigma), stained with ethidiumbromide, and band densitometry readings determined (Gel Doc 2000, BioRad Laboratories, Hercules, CA). Results are expressed as the ratio of banddensities of the target (pre-proenkephalin mRNA) to that of the

Table 1

Serum levels of total bilirubin and alanine aminotransferase (ALT) in bile duct (days 2, 4, 6, 8 and 10 post-surgery) and sham resected mice

Sham Day 2 Day 4 Day 6 Day 8 Day 10

ALT (U/L) 32.8G0.9 65.2G7.4 87.5G21.7* 97.6G11.5** 120.3G11.9*** 93.7G5.3**

Total Bilirubin (mg/dl) 0.25G0.01 1.74G0.38 3.34G0.57 5.42G1.60 24.12G1.25** 32.58G8.34***

Data represent the meanGSEM of results from four mice per group. *P!0.05; **P!0.01 and ***P!0.001 vs sham group.

L. Nelson et al. / Journal of Hepatology 44 (2006) 1141–1149 1143

housekeeping gene (b actin mRNA) and are reported as arbitrarydensitometric units.

(ii) Paw Met-enkephalin Immunohistochemistry: Paw tissues (nZ3mice/group) were removed from formalin, embedded in paraffin, andsectioned (6 m sections). Tissues were then processed for immunohisto-chemistry to detect tissue met-enkephalin expression using a specific met-enkephalin tissue immunohistochemistry staining kit following themanufacturers guidelines (Peninsula Laboratories, Inc., San Carlos, CA).Met-enkephalin was identified using AEC as chromagen (appears as a redcolor with light microscopy).

In additional immunohistochemistry experiments paw tissues fromBDR mice were stained with rat monoclonal antimouse F4/80 antibody(Serotec; stains cells of monocyte/macrophage lineage) followed by arabbit antirat biotinylated secondary antibody (Vector Labs) and DAB(Sigma) as chromagen (appears as reddish brown color under lightmicroscopy).

2.6. Statistical analysis

Data are presented as meanGSEM. For comparisons between twomeans a Student’s unpaired t-test was used. The withdrawal latency andnociceptive score data were analyzed using a one-way analysis of variancefollowed by a Dunnett’s test. With all statistical analyses, P!0.05 wasconsidered significant.

0 2 4 6 8 106

8

10

12

14A

**

Days after surgery

Days after surgery

With

draw

al la

tenc

y (s

)

0 2 4 6 8 100

5

10

15

20

25

30

35 Sham

Cholestatic Filament 4.08

* **

C

Noc

icep

tive

scor

e (%

)

Fig. 1. Effects of cholestasis on thermal (A), or mechanical (B-D) nociception

surgery. Withdrawal latency in response to a thermal stimulus (A) or nocicep

(3.22, 4.08 and 4.17 mN, panels B, C, and D, respectively) were recorded. Dat

surgery) values; P!0.05.

3. Results

3.1. Animal model of cholestasis

Bile duct resected mice developed biochemical evidence

of liver disease and cholestasis as demonstrated by an

increase in serum bilirubin and alanine aminotransferase

levels (Table 1).

3.2. Cholestasis caused increased nociceptice threshold in

response to both thermal and mechanical stimulation

in mice

While sham surgery did not significantly modify

nociceptive response to a thermal stimulus from day 6 to

day 8, the induction of cholestasis significantly increased

the withdrawal latency of mice when their paws were

exposed to a halogen lamp, as compared to basal (before

surgery, time 0) values (Fig. 1A). When a mechanical

Days after surgery

Days after surgery

0 2 4 6 8 100

1

2

3

4

5 Sham

Cholestatic Filament 3.22

B

Noc

icep

tive

scor

e (

%)

0 2 4 6 8 100

10

20

30

40

50 Sham

CholestaticFilament 4.17

D

Noc

icep

tive

scor

e (%

)

in mice at different days after bile duct resection (cholestatic) or sham

tive scores in response to von Frey filaments of different bending forces

a are meanGSEM; *, significantly different from basal (time 0, before

0 1 2 3 4 5 6 77.5

8.5

9.5

10.5

11.5

12.5 saline

nal.

nal. meth.

Sham

A

Days after sham surgery

With

draw

al la

tenc

y (s

)

With

draw

al la

tenc

y (s

)

0 1 2 3 4 5 6 77.5

8.5

9.5

10.5

11.5

12.5 saline

nal.

nal. meth.

Cholestatic

*

**

B

Days after ligation

0 1 2 3 4 5 6 70

5

10

15

20

25

30

35 salinenal.nal. meth.

Sham

Filament 3.84C



Noc

icep

tive

scor

e (%

)

0 1 2 3 4 5 6 70

10

20

30

40 saline

nal.

nal. meth.

Cholestatic

*

Filament 3.84

**

D

Days after ligation

Days after ligation

Noc

icep

tive

scor

e (%

)

0 1 2 3 4 5 6 715

20

25

30

35

40

45

saline

nal.

nal. meth. Sham

Filament 4.08E

Noc

icep

tive

scor

e (%

)

0 1 2 3 4 5 6 70

5

10

15

20

25

30

35

40

45 salinenal.nal. meth.

Cholestatic

Filament 4.08

* *

F

Noc

icep

tive

scor

e (%

)

Fig. 2. Effects of saline, naloxone (nal.) or naloxone methiodide (nal. meth.) treatments on thermal (A, B) and mechanical (C–F) nociception, in sham

control (A, C, E) and cholestatic (B, D, F) mice. Withdrawal latency in response to a thermal stimulus (A, B) or nociceptive scores in response to von

Frey filaments of different bending forces (3.84 mN panels C and D, and 4.08 mN, panels E and F) were recorded. Data are meanGSEM; *,

significantly different from basal (time 0, before surgery) values; P!0.05.


stimulus was applied to the mouse paw (von Frey filaments

of different sizes representing different bending forces) the

nociceptive score of sham animals was not different from

basal values. However, cholestatic mice exhibited decreased

nociceptive score for two filaments of small and intermedi-

ate sizes (Fig. 1B and C). In the case of the small filament

(Fig. 1B), cholestatic mice had no nociceptive reaction

(score of 0 from day 1 to day 10). For the large size filament,

cholestatic mice, like sham controls, did not show

nociceptive scores different from basal values (Fig. 1D).

Taken together, these results show that in mice induction of

cholestasis caused a decreased nociceptive threshold

characteristic of analgesia, in response to both a thermal

and light to moderate mechanical stimuli.

3.3. Involvement of opioid receptors in cholestasis-induced

increased nociceptice threshold

Further, we showed that systemic pre-treatment of mice

with the opioid receptor antagonist naloxone had no effect

Sham BDR0.0

0.5

1.0

1.5

2.0

2.5

3.0

*

A

B

PENK

Actin

sham BDR

Pre

-pro

en

kep

ha

linm

RN

A le

vels

(a

s a

ra

tioo

f β

Act

in m

RN

A le

vels

;A

rbitr

ary

De

nsi

tom

etr

icU

nits

)

Fig. 4. Pre-proenkephalin mRNA expression in paw tissues obtained

from cholestatic (BDR) and sham control mice 10 days post-surgery.

(A) Bars represent meanGSEM of data (pre-proenkephalin mRNA

levels expressed as a ratio of b actin housekeeping gene levels) from 4

sham and 5 BDR mice; P%0.05. (B) Representative ethidium bromide

stained agarose gel of RT-PCR products of paws obtained from 2 BDR

and 2 sham mice (PENKZpre-proenkephalin; b actinZhousekeeping

gene).


on withdrawal latency in response to a thermal stimulus in

sham mice (Fig. 2A), but significantly inhibited thermal

antinociception in cholestatic mice as compared to saline-

treated mice (Fig. 2B). Similarly, naloxone treatment did

not modify nociceptive scores in response to a mechanical

stimulus (von frey filaments 3.84 and 4.08) in sham-

operated mice (Fig. 2C and E). However, naloxone

treatment inhibited the decreased nociceptive score

observed for those two filaments in cholestatic mice,

compared to basal values (Fig. 2D and F).

3.4. Peripheral activation of opioid receptors is responsible

for cholestasis-induced increased nociceptive threshold

In order to investigate whether the antinociceptive effects

of cholestatis involved a peripheral or a central opioid

receptor mechanism, we used an opioid receptor antagonist

that does not cross the blood–brain-barrier and acts only at a

peripheral level: naloxone methiodide [26]. Systemic

treatment of mice with naloxone methiodide did not modify

nociceptive responses to thermal or mechanical stimulation

in sham-operated mice (Fig. 2A, C, and E). In cholestatic

mice treated systemically with naloxone methiodide, a

similar profile as the one observed after naloxone treatment

was shown: naloxone methiodide was able to completely

inhibit cholestasis-induced increased withdrawal latency

(Fig. 2B), and cholestatis-induced decreased nociceptive

score (Fig. 2D, and F). To further show that the effects of

naloxone methiodide were truly peripheral, we injected very

Basal Saline 0.5 h Nal. Met. 2 h Nal. Met.7

8

9

10

11ShamCholestatic (7days)*

° °

A

With

draw

al L

aten

cy (

s)

Basal Saline 0.5 h Nal. Met. 2 h Nal. Met.10

20

30

40

50 ShamCholestatic °

°

*

B

Noc

icep

tive

scor

e (%

)

Fig. 3. Effects of intraplantar injection of naloxone methiodide (Nal.

Met.) 30-min and 2-h after its administration, or saline intraplantar

injection, on withdrawal latency in response to a thermal stimulus (A),

or mechanical von Frey (4.08 mN) stimulus (B), in sham and cholestatic

mice, 7-days after surgery. Data are meanGSEM; *, significantly

different from basal (time 0, before surgery) values; 8, significantly

different from saline P!0.05.

low dose naloxone methiodide directly into the mouse paw

and determined nociceptive responses to thermal and

mechanical stimuli in sham and cholestatic mice, 7-days

after surgery (the time-point for, which the strongest

antinociceptive effect was observed, as shown in Fig. 1).

Our results show that intraplantar injection of naloxone

methiodide was able to significantly reduce cholestasis-

induced increased withdrawal latency 30-min, and 2-h after

the injection, but had no effect on thermal nociception of

sham-operated mice (Fig. 3A). After mechanical stimu-

lation (von Frey filament 4.08), local injection of naloxone

methiodide did not modify nociceptive score in sham, but

significantly reduced the antinociceptive effects observed in

cholestatic mice (Fig. 3B).

3.5. Increased plantar met-enkephalin mRNA and protein

expression in cholestasis

Proenkephalin represents the precursor protein of met-

enkephalin and is cleaved after synthesis to give met-

enkephalin. We observed a significant 1.4-fold increase in

paw tissue pre-proenkephalin mRNA levels in BDR

compared to sham mice (Fig. 4A and B). By immunohis-

tochemistry, BDR and sham mice demonstrated significant,

but similar levels of expression of met-enkephalin in cells

contained within the peri-follicular apparatus within the

paw dermis (Fig. 5A and C). In contrast, located within

Fig. 5. Immunohistochemistry for met-enkephalin (stained bright red) in paw tissues obtained from sham (A and B) and BDR (C and D) mice 10 days

post-surgery. Filled black arrows point to met-enkephalin positively staining cells within the peri-follicular apparatus in the paws of BDR and sham

mice. Open arrows point to large macrophage-like cells in the dermis of BDR mice, which stain strongly for met-enkephalin. These large cells within

the dermis of BDR mice stain positively for F4/80, a marker for monocytes/macrophages (E; stained reddish-brown color). All pictures were taken at

40! power (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article.).


the dermis of only BDR mice were large cells, which stained

strongly for met-enkephalin expression (Fig. 5B and D) and,

which also stained positively for the monocyte/macrophage

marker F4/80 (Fig. 5E).

4. Discussion

The current study confirms previous experimental

observations indicating that cholestasis is associated with

naloxone reversible antinociception [15]; however our

current findings expand on previous observations and

demonstrate that cholestasis-associated antinociception is

elicited by using both mechanical and thermal nociceptive

stimuli. Peripherally administered naloxone readily crosses

the blood–brain-barrier and therefore blocks opioid

receptors on both peripheral nerves and within the CNS.

The blockade of cholestasis associated antinociception had

been attributed to a central effect of naloxone [12,15];

however, our results clearly demonstrate that cholestasis-

induced antinociception results from activation of opioid

receptors outside of the CNS. More specifically, our data

strongly suggests that endogenous opioids act at cutaneous

pain carrying nerve fiber endings in cholestasis to induce

antinociception. Furthermore, we have documented that

cholestasis is associated with the increased cutaneous

production of at least one endogenous opioid (i.e. met-

enkephalin). This is a novel finding with potential clinical

implications for both understanding the pathogenesis of

cholestasis associated pruritus as well as in its management.

Pruritus is a poorly understood sensation, which results

classically from a pruritogen activating specific itch-

dedicated unmyelinated cutaneous C-fiber nerve endings

[3,5,8,9]. The pruritogen(s) involved in cholestasis associ-

ated itch are poorly defined. However, both bile acids and

histamine, which accumulate in the circulation in choles-

tasis [16,17], are capable of activating cutaneous itch fibers

[3,9,27]. The bile salt binding resin cholestyramine has been

Fig. 6. Schematic drawing demonstrating the activation of cutaneous

itch fibers by an unknown pruritogen (e.g. bile acids) in cholestatic

mice, which increases activity in spinal itch pathways. In parallel,

activity in cutaneous pain fibers is suppressed by endogenous opioids

(e.g. met-enkephalin; M-enk) produced by macrophages within the

dermis, resulting in decreased activity in spinal pain pathways, which

leads to a reduction in the suppressive effect, which spinal pain

processing neurons normally exert upon spinal itch processing

neurons. The result is an augmentation in the activity of spinal itch

processing pathways and increased pruritus.


used clinically to effectively treat cholestasis associated

pruritus for decades consistent with a role for bile salts in the

generation of cholestatic pruritus [1]. The role for histamine

in cholestasis associated itch is more questionable as

antihistamines appear to be minimally effective in chole-

static pruritus [1].

Opiates are widely used clinically to relieve pain [7].

However, a common side effect of both peripheral and

centrally administered opiates (e.g. morphine) is pruritus

[7,28]. Opiate-induced pruritus is poorly understood but

may involve drug actions at a number of sites. Within the

CNS, an ‘itch center’ has been postulated as lying within the

medulla oblongata [29]. However, activation of this center

by central opiates results typically in pruritus confined to the

nose and upper part of the face [30]. This pattern of itching

is uncommonly observed in cholestatic patients.

Recently, opioid receptors have been identified on

cutaneous nerve endings [18,19]. Activation of these

receptors by opiates or endogenous opioids results in

antinociception [18,19,31], an effect blocked by local

peripheral application of opioid receptor antagonists [31].

Stimulation of cutaneous nerve pain fibers by opioids leads

to decreased activity within these nerves and ultimately

decreased pain processing activity within neurons in the

spinal dorsal horn [8,9,32] (Fig. 6). This effect could then

lead to an increase in pruritus through a decrease in

inhibitory activity of spinal itch processing neurons.

Elevated levels of circulating endogenous opioids have

been identified in patients and experimental animals with

cholestasis [10,11]. Moreover, rodents with experimental

cholestasis exhibit naloxone-reversible antinociception

[15], an observation confirmed and expanded upon in the

current study. These findings would be consistent with

enhanced opioid driven attenuation of pain processing

pathways in cholestatic rodents. However, the specific site

where endogenous opioids are producing this antinocicep-

tive effect (i.e. within or outside the CNS) is not known. Our

current results demonstrate that opioid-mediated antinoci-

ception in cholestasis is the result of endogenous opioid

effects outside of the CNS. However, opioids can inhibit

pain fiber nerve transmission at two main locations when

acting upon pain impulse carrying nerves outside of the

CNS; namely at the level of the cutaneous nociceptor or at

the level of the nerve cell body within the dorsal root

ganglion [33]. Our findings indicating a reversal of

cholestasis-associated antinociception by the intraplantar

injection of a very low dose of naloxone methiodide (i.e.

only blocking receptors on peripheral nerve endings)

strongly suggest that the effects of endogenous opioids in

cholestasis associated antinociception are occurring at the

level of these cutaneous nociceptors. Moreover, these

results may have direct implications for cholestasis

associated pruritus, and may also explain why scratching

does not typically alleviate cholestasis associated itch [1,2].

Interestingly, endogenous opioids can augment histamine-

induced cutaneous itch in normal volunteers [34],

suggesting a possible indirect effect of endogenous opioids

in the initiation of cholestasis associated itch via a

mechanism similar to that outlined in Fig. 6.

Our suggestion that cholestasis-associated antinocicep-

tion occurs at the level of cutaneous nerve endings is further

supported by our findings documenting an increase in the

local plantar (i.e. dermal) expression of the endogenous

opioid, met-enkephalin, in cholestatic compared to control

mice. We specifically examined the pattern of tissue met-

enkephalin expression in these mice as circulating met-

enkephalin levels are known to be elevated in cholestatic

patients and have been implicated in cholestasis associated

itch [10]. We have identified that BDR mice exhibit an

influx of macrophage-type cells (i.e. F4/80Cve) into the

paw dermis and these cells appear to be the cutaneous

source of met-enkephalin production in these cholestatic

mice. Moreover, monocytes/macrophages can be important

sources of met-enkephalin production in the skin [35,36].

Consistent with this observation, paw pre-proenkephalin

mRNA levels were significantly increased in BDR vs sham

mice. Our demonstration of increased pre-proenkephalin

mRNA levels in the paws of cholestatic mice does not

appear to be simply a non-specific effect of cholestasis as

pro-opiomelanocortin (POMC) mRNA levels are signifi-

cantly reduced in the paws of BDR compared to sham mice

(MG Swain, unpublished observation; POMC is the

precursor for another endogenous opioid, b endorphin).

Our current observations that antinociception in choles-

tasis is due to opioid effects outside of the CNS does not


preclude other actions of endogenous opioids within the

CNS during cholestasis. Opioid withdrawal-like symptoms

experienced by some pruritic cholestatic patients treated

with opioid receptor antagonists strongly implicate

enhanced central release of endogenous opioids within the

cortical regions of the CNS in these patients [13,14].

However, it is unclear whether the endogenous opioids

acting at these higher CNS centers mediate itch (at least in

part) or modulate the perception of itch.

In conclusion, we have identified that cholestasis-

associated antinociception is mediated by endogenous

opioids acting at the level of cutaneous pain fiber nerve

endings. These results may have direct relevance to

cholestasis associated pruritus and support clinical trials in

using opioid receptor blockers, which do not enter the CNS

to treat patients with cholestasis-induced itch, thereby

avoiding unwanted or clinically problematic CNS side

effects of opioid receptor blockade in cholestatic patients

[13,14,37].

Acknowledgements

This work was supported by operating grants from the

CIHR (MGS and NV). MGS is an AHFMR Senior Scholar

and a CIHR Investigator and NV is an AHFMR Scholar and

CIHR New Investigator.

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Endogenous opioid-mediated antinociception in cholestatic mice is peripherally, not centrally,...

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