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The Journal of Neuroscience, July 15, 2001, 21(14):5281-5288
Inhibition of Neuropathic Pain by Selective Ablation of Brainstem
Medullary Cells Expressing the µ-Opioid Receptor
Frank
Porreca1,
Shannon
E.
Burgess1,
Luis R.
Gardell1,
Todd W.
Vanderah1,
T. Philip
Malan Jr1,
Michael H.
Ossipov1,
Douglas A.
Lappi2, and
Josephine
Lai1
1 Departments of Pharmacology and Anesthesiology,
University of Arizona, Tucson, Arizona 85724, and
2 Advanced Targeting Systems, San Diego, California 92121
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ABSTRACT |
Neurons in the rostroventromedial medulla (RVM) project to spinal
loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a
mechanism of chronic pain. This possibility and the phenotype of RVM
cells that might underlie experimental neuropathic pain were
investigated. Cells expressing µ-opioid receptors were targeted with
a single microinjection of saporin conjugated to the µ-opioid agonist
dermorphin; unconjugated saporin and dermorphin were used as controls.
RVM dermorphin-saporin, but not dermorphin or saporin, significantly
decreased cells expressing µ-opioid receptor transcript. RVM
dermorphin, saporin, or dermorphin-saporin did not change baseline
hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve
ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin
failed to elicit the expected increase in sensitivity to non-noxious
mechanical or noxious thermal stimuli applied to the paw. RVM
dermorphin or saporin did not alter SNL-induced experimental pain, and
no pretreatment affected the responses of sham-operated groups. This
protective effect of dermorphin-saporin against SNL-induced pain was
blocked by  -funaltrexamine, a selective µ-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with
the µ-opioid receptor. RVM microinjection of dermorphin-saporin, but
not of dermorphin or saporin, in animals previously undergoing SNL
showed a time-related reversal of the SNL-induced experimental pain to
preinjury baseline levels. Thus, loss of RVM µ receptor-expressing cells both prevents and reverses experimental neuropathic pain. The
data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.
Key words:
neuropathic pain; descending facilitation; RVM; µ-opioid receptors; saporin; ON cells
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INTRODUCTION |
The presence of endogenous
pain-modulating mechanisms is well established. Although pain
inhibitory systems projecting from the brainstem to the spinal cord
offer obvious survival advantages, the role of descending projections
that facilitate pain transmission is less clear (Watkins and Mayer,
1982 ; Wiertelak et al., 1992; Mason et al., 1999). Nevertheless, the
presence of pain facilitation mechanisms raises the possibility that
their abnormal sustained activity may underlie chronic pain.
Injury to nerves may elicit neuropathic pain characterized, in part, by
increased sensitivity to normally non-noxious and noxious stimuli
(allodynia and hyperalgesia, respectively) (Payne, 1986; Chaplan and
Sorkin, 1997). Aspects of this human condition have been modeled by
injury to spinal nerves in animals (Bennett and Xie, 1988; Seltzer et
al., 1990; Kim and Chung, 1992; Kim et al., 1997). Increased
spontaneous and persistent afferent discharge may be critical in
eliciting hypersensitivity of spinal neurons (i.e., central
sensitization) (Kirk, 1974; Wall and Gutnick, 1974; Devor, 1991, 1994;
Kajander et al., 1992). Both injured as well as adjacent uninjured
fibers become spontaneously active after injury (Li et al., 2000; Wu et
al., 2001). Manipulations designed to interfere with ascending,
large-fiber projections to brainstem nuclei, including spinal
transection, ipsilateral and contralateral hemisections, and selective
lesions of the ipsilateral or contralateral (relative to the side of
peripheral nerve injury) dorsal columns block nerve injury-induced pain
(Bian et al., 1998; Sung et al., 1998; Sun et al., 2001). Lidocaine
injection into the ipsilateral (relative to the side of peripheral
nerve injury), but not contralateral, nucleus gracilis also blocks
nerve injury-induced pain (Sun et al., 2001). These studies support a
role for central processes in the mediation of experimental neuropathic pain.
Blockade of established nerve injury-induced pain is also produced by
lidocaine microinjection into the rostral ventromedial medulla (RVM),
indicating additionally the importance of descending modulatory
pathways (Pertovaara et al., 1996; Kovelowski et al., 2000) and the
possibility of tonic discharge of cells mediating descending
facilitation (Fields et al., 1991; Fields, 1992). The importance of
descending facilitation is also supported by observations that lesions
of the dorsolateral funiculus (DLF) block nerve injury-induced pain
(Ossipov et al., 2000). One characteristic of RVM cells that may
mediate descending facilitation is their sensitivity to opioid µ receptor agonists (Fields et al., 1983; Fields and Heinricher, 1989;
Pan et al., 1990; Heinricher et al., 1994). These findings suggest that
RVM cells that may mediate descending facilitation in chronic pain
states might be identified by their expression of this opioid receptor.
The present experiments tested this hypothesis by targeting RVM µ receptor-expressing cells with the cytotoxin saporin, by conjugating
the toxin to a potent opioid µ receptor agonist, dermorphin (Broccardo et al., 1981; Braga et al., 1984). The goal was to determine
the role of RVM µ-opioid receptor-expressing cells in preventing or
reversing nerve injury-induced pain. A similar approach has been
successfully used to lesion spinal cord lamina I projection cells with
a substance P-saporin conjugate (Mantyh et al., 1997; Nichols et al.,
1999).
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MATERIALS AND METHODS |
Radioligand binding. Radioligand binding was
performed using crude membrane preparations from NG 108-15 (expresses
mouse opioid  receptors) and from transfected cells that express the
rat opioid µ receptors (MORs). All radioligand binding assays were
performed in duplicate in 50 mM Tris, pH 7.4, in
the presence of 0.5 mg/ml bovine serum albumin (BSA) and protease
inhibitors (30 µM bestatin, 10 µM captopril, 0.37 U/ml bacitracin, and 0.1 mM phenylmethylsulfonyl fluoride). All
reactions were performed at 25°C for 3 hr in a total reaction volume
of 1 ml. At least 10 concentrations of dermorphin (10 14 to
105
M) or dermorphin-saporin
(1014 to
107.5
M) were used. The concentration of
3H-[D-Ala2,
NMPhe4, Gly-015]enkephalin (2.2 nM) was based on the
Kd value of the radioligand determined
from saturation analysis. The reaction was terminated by rapid
filtration through Whatman GF/B filters presoaked in polyethyleneimine
and washed three times with 4 ml of ice-cold PBS. Nonspecific binding
was defined as that in the presence of 10 µM
naloxone. Radioactivity in the samples was determined by liquid
scintillation counting. Data were analyzed by nonlinear regression
analysis using GraphPad Prism (Graph Pad, San Diego, CA). The
Ki value(s) for each ligand was
calculated from the IC50 value(s) based on the
Cheng and Prusoff equation from at least three independent experiments.
Intracranial drug microinjection. All rats were prepared for
bilateral RVM drug administration as we have described previously (Kovelowski et al., 2000). Anesthetized (ketamine or xylazine, 100 mg/kg, i.p.) animals were placed in a stereotaxic head holder. For
intracranial bilateral drug administrations, the skull was exposed, and
two 26 ga guide cannulas separated by 1.2 mm (Plastics One Inc.,
Roanoke, VA) were directed toward the lateral portions of the RVM
(anteroposterior,  2.0 mm; dorsoventral, 0 mm; and lateral,
±0.6 mm from stereotaxic zero based on the intra-aural line). The
guide cannulas were secured to the skull, and the animals were allowed
to recover for 5 d after surgery before any drug administration.
Drug administrations into the RVM were performed by slowly expelling
0.5 µl of drug solution through a 33 ga injection cannula inserted
through the guide cannula and protruding an additional 1 mm into fresh
brain tissue. Dermorphin, saporin, or dermorphin-saporin was
administered as a single dose of 3 pmol into the RVM (1.5 pmol in 0.5 µl on each side).
Antinociceptive testing. Response thresholds to innocuous
mechanical stimuli were evaluated by determination of paw withdrawal after probing of the paw with a series of calibrated von Frey filaments. Each filament was applied perpendicularly to the plantar surface of the paw, ipsilateral to the nerve injury, of rats kept in
suspended wire-mesh cages. The withdrawal threshold was determined by
sequentially increasing and decreasing the stimulus strength ("up and
down" method), analyzed using a Dixon nonparametric test (Dixon,
1980). Data are expressed as the mean withdrawal threshold. Response
thresholds to noxious thermal stimuli were evaluated by determination
of paw withdrawal from a focused beam of radiant heat. Rats were
acclimated within Plexiglas enclosures on a clear glass plate, and a
radiant heat source was directed onto the plantar surface of the
hindpaw. Paw-withdrawal latency was determined by a motion detector.
The latency to withdrawal of the paw from the radiant heat source was
determined both before and after drug or vehicle administration. A
maximal cutoff of 40 sec was used to prevent tissue damage. The
tail-flick test was performed by determining latency to withdrawal from
a 52°C water bath. Data are expressed as percentage of maximal
possible effect (% MPE), which is 100 × (test baseline)/(15 baseline). A 15 sec cutoff was used.
Spinal nerve injury. Spinal nerve ligation (SNL) injury was
induced using the procedure of Kim and Chung (1992). Male Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN; 200-300 gm) were
maintained in a climate-controlled room on a 12 hr light/dark cycle and
with access to food and water ad libitum. Surgery was performed in accordance with the policies and recommendations of the
National Institutes of Health guidelines for the handling and use of
laboratory animals and was approved by the Institutional Animal Care
and Use Committee of the University of Arizona. Rats were anesthetized
with halothane vaporized in 95% O2/5%
CO2 "to effect." After surgical preparation
of the rats and exposure of the dorsal vertebral column from L4 to S2,
the exposed L5 and L6 spinal nerves were tightly ligated with 4-0 silk
suture. The incision was closed, and the animals were allowed to
recover for 5 d. Rats that exhibited motor deficiency or failure
to exhibit subsequent increased sensitivity to innocuous mechanical
stimulation were excluded from further testing (<5% of the animals
were not used). Sham control rats underwent the same operation and
handling as the experimental animals, but without SNL. Evaluation of
response thresholds was performed on the hindpaw ipsilateral to SNL or sham SNL using the procedures described above.
In situ hybridization of opioid µ receptor
mRNA. For tissue preparation, male Sprague Dawley rats were deeply
anesthetized with ketamine and perfused transcardially with PBS treated
with 0.1% diethylpyrocarbonate (DEPC), followed by 4%
paraformaldehyde. Whole rat brains were removed and post-fixed in
fixative overnight, cryoprotected in 30% sucrose in PBS treated with
0.1% DEPC, and stored at 4°C. Frozen frontal sections (20-40 µm)
were prepared from the brainstem caudal to the site of incision of the
cannulas and mounted on positively charged slides. A single-stranded,
fluorescein-labeled partial cDNA probe corresponding to nucleotides
628-965 of the coding region of the rat opioid µ receptor cDNA was
synthesized by PCR by use of a 100:1 ratio of antisense
(3')-to-sense (5') primers and a mixture of fluorescein-labeled dUTP
and unlabeled dNTP. The PCR was performed for 30 cycles (45 sec at
95°C, 60 sec at 60°C, and 2 min at 72°C); the product was
purified by ethanol precipitation. The probe was reconstituted in
RNase-free 1× Tris-EDTA and analyzed by agarose gel
electrophoresis and Southern transfer to determine the yield. A
corresponding sense probe was synthesized under the same conditions
except using a 1:100 ratio of antisense-to-sense primers, with or
without fluorescein-dUTP. In situ hybridization was
performed using both standard and proprietary reagents from InnoGenex
(San Ramon, CA) under conditions that were carefully optimized for our
cDNA probe. Briefly, mounted tissue sections were post-fixed with 1%
formaldehyde and then deproteinated with proteinase K (100 µg/ml).
After a second 1% formaldehyde treatment, the sections were heated to
80°C for 5 min in a hybridization buffer containing formamide,
dextran sulfate, and the fluorescein-labeled probe (typically 1:2 to
1:5 dilution) and allowed to cool to 37°C, and the hybridization was
continued for 16 hr at 37°C. The sections were washed extensively
with PBS and 0.1% Tween 20, preblocked with a buffer containing casein
and sodium azide, and then incubated with a biotinylated
anti-fluorescein antibody. The sections were washed, followed by
incubation with a 1:3 dilution of streptavidin-alkaline phosphatase
conjugate. The sections were developed by incubating with fast red for
1 hr, counterstained with Mayer's hematoxylin, and mounted with
SuperMount. RNase pretreatment was performed by incubating sections
with 200 µg/ml RNase A in 100 mM Tris, pH 8.0, and 0.5 M NaCl for 60 min at 37°C, followed by
extensive washing before hybridization. Computer-assisted mapping was
performed under bright-field illumination of coronal sections using an
image-combining computer microscope using Neurolucida software
(Microbrightfield Inc., Baltimore, MD). The boundaries of the facial
nuclei and pyramidal tracts were manually traced using a Nikon 4×
objective. The sections were then systematically scanned for
labeled neurons using a Merzhauser motorized stage and a Nikon 40×
objective. Bright-field images of tissue sections were acquired with a
Nikon E800 microscope outfitted with a Hamamatsu C5810 color CCD camera and a 40× plan fluor 0.75 numerical aperture objective lens. The digitized output of the camera was acquired with a microcomputer.
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RESULTS |
Binding affinity
The radioligand binding studies in membrane preparations from NG
108-15 transfected cells that express the rat opioid µ receptors demonstrated that the conjugation of saporin to dermorphin did not
significantly alter the affinity of dermorphin for opioid µ receptors. As expected, dermorphin alone demonstrated a high affinity
for the µ-opioid receptor and a Ki
value of 0.7 nM. The Ki value for dermorphin-saporin was
0.1 nM.
Antinociceptive activity
The bilateral microinjection of 3 pmol of dermorphin or of
dermorphin-saporin directly into the RVM produced a robust
antinociceptive effect in the 52°C hot-water tail-flick test. The
peak antinociceptive effect of dermorphin, 78 ± 13.2%
MPE, was not significantly different from that of the
dermorphin-saporin conjugate, which was 59 ± 4.7% MPE
(p > 0.5, Student's t test). The
microinjection of unconjugated saporin into the RVM did not elicit any
changes in tail-flick latency.
Prevention of SNL-induced neuropathic pain
The bilateral microinjection of 3 pmol of dermorphin, saporin, or
dermorphin-saporin into the RVM of naive rats produced no observable
behavioral changes over a period of 28 d. Paw withdrawal thresholds to probing with von Frey filaments remained unchanged over
this time period (Fig.
1A). Similarly, the paw
withdrawal latencies to radiant heat applied to the plantar aspect of
the hindpaw did not change over this 28 d period (Fig.
1B). Furthermore, the nociceptive tail-flick reflex
to 52°C water immersion was also unaffected in these experimental
animals (Fig. 1C).
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Figure 1.
Male Sprague Dawley rats received a bilateral
microinjection of saporin, dermorphin, or dermorphin-saporin into the
RVM. A, B, The animals were tested for responses to
non-noxious mechanical stimuli (von Frey filaments)
(A) and to noxious radiant heat
(B) on days 0 (baseline), 2, 7, 14, and 28 after
the microinjection. No significant changes
(p > 0.05) were observed in any of the
groups of rats over this observation period (ANOVA). C,
The response latency to noxious heat applied to the tail (tail-flick
reflex using a 52°C water bath) was also determined before
dermorphin-saporin and again at day 28 after the microinjection; no
change in response latencies was observed. n = 6 rats per group.
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The same group of rats then underwent either sham or SNL surgery and
was tested again 7 d after surgery. The rats that initially received either saporin or dermorphin alone demonstrated a clear development of behavioral signs of neuropathic pain associated with SNL
(Fig. 2). Rats receiving SNL and either
RVM dermorphin or saporin alone displayed tactile allodynia and thermal
hyperalgesia as evidenced by a significant reduction in paw withdrawal
thresholds (Fig. 2A). Likewise, the paw withdrawal
latencies for the dermorphin- and saporin-pretreated groups were
significantly (p  0.05) decreased 7 d
after SNL (Fig. 2B). In contrast, the
dermorphin-saporin-pretreated SNL group showed responses to
non-noxious or noxious stimuli that did not differ significantly from
the pre-SNL baseline values in response either to probing with von Frey
filaments or to noxious radiant heat (Fig. 2). The responses of the
sham-operated rats did not demonstrate any evidence of neuropathic pain
behavior with either probing with von Frey filaments or noxious radiant heat.
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Figure 2.
Male Sprague-Dawley rats received a bilateral
microinjection of saporin, dermorphin, or dermorphin-saporin into the
RVM. After 28 d, rats were subjected to either SNL or sham
surgery. Seven days after surgery, the animals were tested for their
responsivity to normally non-noxious mechanical
(A) or noxious thermal (B)
stimuli applied to the paw ipsilateral to the sham or SNL procedure.
Rats that had received RVM pretreatment of either saporin or dermorphin
demonstrated increased sensitivity to both normally non-noxious
mechanical and noxious thermal stimuli after SNL, as indicated by the
significantly (*p 0.05, Student's
t test) decreased thresholds to these stimuli when
compared with the pretreatment baseline levels. However, rats with SNL
that were pretreated with RVM dermorphin-saporin showed response
thresholds to mechanical or thermal stimuli that were not significantly
different from baseline values (p > 0.05).
None of the sham SNL groups demonstrated any appreciable changes in
behavioral responses when compared with the pretreatment baseline after
any RVM pretreatment.
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Prevention of neuropathic pain
The paw withdrawal thresholds to probing with von Frey filaments
and the latencies to withdrawal from noxious radiant heat were
determined before any manipulations. The pooled paw withdrawal thresholds were 12.6 ± 0.88 gm, and the pooled paw withdrawal latencies were 20 ± 1.15 sec (Fig.
3). Rats with either SNL or sham surgery
were randomly separated into three groups each destined for
microinjection of dermorphin, saporin, or dermorphin-saporin into the
RVM. Rats with SNL showed clear behavioral signs of neuropathic pain on
the fifth day after surgery. Tactile allodynia was indicated by the
significant (p 0.05) reductions in the paw
withdrawal thresholds of rats with SNL to innocuous mechanical light
touch (Fig. 3A). Thermal hyperalgesia was indicated in rats
with SNL by the significant (p 0.05)
reduction in paw withdrawal latencies to noxious radiant heat (Fig.
3B). After these baseline determinations were made, rats
received 3 pmol of dermorphin, saporin, or dermorphin-saporin bilaterally into the RVM. Animals were tested at 2, 7, 14, and 28 d after the microinjections. Rats with SNL that received the dermorphin-saporin conjugate demonstrated a gradual loss of heightened sensitivity to both innocuous mechanical and noxious thermal stimuli over the 28 d observation period (Fig. 3). On day 28, the paw withdrawal thresholds to probing with von Frey filaments were significantly (p 0.05) elevated and not
significantly different (p > 0.05) from
preligation values (Fig. 3A) in rats with SNL. Similarly,
paw withdrawal latencies to noxious radiant heat were not significantly
(p 0.05) different from pre-SNL values
28 d after the microinjection of dermorphin-saporin (Fig.
3B). The microinjection of 3 pmol of either dermorphin or
saporin alone did not effect any changes in the behavioral responses to
light tactile or noxious thermal stimuli. On day 28 after the
microinjection of dermorphin or saporin into the RVM, the paw
withdrawal thresholds and latencies of the rats with SNL were
essentially unchanged and significantly lower than pre-SNL values
(p > 0.05) (Fig. 3).
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Figure 3.
Baseline responses to von Frey filaments and to
noxious radiant heat were determined in male Sprague Dawley rats. The
rats were then subjected to SNL. After increased sensitivity to
normally non-noxious mechanical (A) and noxious
thermal (B) stimuli was clearly established in
the SNL rats, they were divided into equal groups and received RVM
microinjections of saporin, dermorphin, or dermorphin-saporin. The
animals were tested for the response threshold to normally non-noxious
mechanical (A) and noxious thermal stimuli
(B) over a 28 d period. By approximately day
2 after the injection, all groups of SNL rats demonstrated clearly
increased sensitivity to normally non-noxious mechanical and noxious
thermal stimuli, as indicated by significantly reduced behavioral
response thresholds compared with the pretreatment baseline levels
(p 0.05, Student's t
test). By postinjection day 8, however, and continuing to the end of
the experiment at day 28, the SNL rats that received an RVM
microinjection of dermorphin-saporin exhibited a progressive reversal
of both mechanical (A) and thermal
(B) thresholds to levels that did not differ
significantly from baseline (p > 0.05). SNL
rats that received dermorphin or saporin retained both behavioral signs
of experimental neuropathic pain. n = 6 rats per
group.
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Blockade with -funaltrexamine
Groups of six male Sprague Dawley rats were tested for acute
antinociception using the 52°C warm-water tail-flick test after RVM
dermorphin (3 pmol), U69,593 (60 nmol), or
[D-Ala2,
Glu4]deltorphin (60 nmol). These opioids
produced an acute antinociceptive effect of 95 ± 4.4, 54 ± 14, or 63 ± 9% MPE, respectively. The rats were allowed to rest
for 7 d, and each then was given an injection of an opioid µ receptor-selective dose of 18.8 nmol of -funaltrexamine (-FNA)
bilaterally (9.4 nmol in 0.5 µl on each side) into the RVM. Each
group of rats was then tested 24 hr later for acute antinociception
with the same dose of receptor-selective opioid as before. Pretreatment
with -FNA did not affect baseline latencies to tail flick when
measured 24 hr after administration but significantly inhibited the
acute antinociception of RVM dermorphin as indicated by an
antinociceptive index of only 9.7 ± 5.6% MPE. In contrast, the
antinociceptive effects of RVM U69,593 or of RVM
[D-Ala2,
Glu4]deltorphin were not changed by
pretreatment with -FNA. The antinociceptive indices obtained were
48 ± 8.8 and 61 ± 10% MPE after -FNA, respectively, values not significantly different from control
(p > 0.05). These data ensure that the dose of
-FNA used was selective for the µ-opioid but not the  - or
-opioid receptors.
The same dose, 18.8 nmol of -FNA, was microinjected bilaterally into
the RVM of naive rats. After 24 hr, the rats received RVM
microinjections of 3 pmol of dermorphin, saporin, or
dermorphin-saporin conjugate. After 28 d, the animals were
subjected to SNL and evaluated for responses to innocuous mechanical
and noxious thermal stimuli 7 d later. The groups that received
-FNA pretreatment before saporin, dermorphin, or dermorphin-saporin
all exhibited increased sensitivity to innocuous mechanical (Fig.
4A) and noxious thermal (Fig. 4B) stimuli as expected with SNL. In contrast,
SNL rats that received saline before the dermorphin-saporin conjugate
did not show any behavioral signs of neuropathic pain. The paw
withdrawal thresholds to von Frey filaments (Fig. 4A)
and the latencies to noxious radiant heat (Fig. 4B)
were not significantly different from the pretreatment baseline
values.
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Figure 4.
Male Sprague Dawley rats were tested for threshold
responses to non-noxious mechanical or noxious thermal stimuli
(baseline values) and subsequently received either the noncompetitive
opioid µ receptor antagonist -FNA or saline into the RVM 24 hr
before the administration of saporin, dermorphin, or
dermorphin-saporin into the RVM. All groups were subjected to SNL
28 d after RVM dermorphin, saporin, or dermorphin-saporin and
tested for their response thresholds to non-noxious mechanical or
noxious thermal stimuli 7 d after SNL surgery. Pretreatment with
-FNA before RVM dermorphin-saporin abolished the ability of RVM
dermorphin-saporin to prevent SNL-induced increased sensitivity to
non-noxious mechanical and noxious thermal stimuli but did not alter
the effects of SNL in saporin- or dermorphin-pretreated rats.
n = 6 rats per group. BL, Baseline;
DERM, dermorphin; SAP, saporin.
*p 0.05 from baseline;  p 0.05 from
SNL.
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In situ hybridization
Our initial analysis using coronal sections from the brainstem
region of naive rats (taken between 11.0 and 13.2 mm from bregma)
showed that the localization of cells expressing µ-opioid receptor
transcripts in the caudal brainstem including the RVM was highly
consistent with that reported previously based on autoradiography for
the receptor or on in situ hybridization for message (Bowker and Dilts, 1988; Bowker et al., 1988; Mansour et al., 1994a,b; Peckys
and Landwehrmeyer, 1999). In the RVM, cells that expressed opioid µ receptor transcripts were highly localized to the raphe magnus and the
magnocellular reticular nucleus and also found at a lower cell density
in the paragigantocellular reticular nucleus-lateral part and the
gigantocellular reticular nucleus. Caudal to the RVM, µ receptor
transcripts were highly localized to cells in the raphe obscurus, the
raphe pallidus, the inferior olivary complex, the external nucleus
cuneatus, the medial solitary nucleus, the hypoglossal nucleus, the
dorsal motor nucleus of the vagus, the spinal trigeminal tract, and the
nucleus ambiguus. A low density of discrete cell bodies in the nucleus
gigantoreticular nucleus was also labeled. The localization of the
cells that express µ receptor mRNA is consistent with that described
previously. For the experiments, coronal sections 20 µm thick were
obtained 28 d after the bilateral microinjection of dermorphin,
saporin, or dermorphin-saporin into the RVM. The brain slices obtained
from rats that had been pretreated with dermorphin or saporin alone showed similar densities of labeling for mRNA of the µ-opioid receptor in the RVM (Fig. 5). The RVM of
the dermorphin-saporin-pretreated rats consists of significantly fewer
labeled cells when compared with that of dermorphin- or
saporin-pretreated rats (Fig. 5). This regional loss of µ receptor
transcripts in dermorphin-saporin-pretreated tissues was not caused by
differences in experimental conditions because the processing of
tissues and the in situ hybridization procedures were
performed in parallel with tissues from dermorphin- or
saporin-pretreated rats. Furthermore, brainstem sections caudal to the
RVM (between 12.2 and 13.2 mm from bregma) taken from dermorphin-,
saporin-, or dermorphin-saporin-pretreated animals showed similar
distributions and densities of labeled cells, suggesting that the loss
of labeling correlates with the stereotaxic delivery of
dermorphin-saporin to the RVM and that this loss is specific to
dermorphin-saporin treatment (data not shown). The immunolabeling could be abolished by pretreatment of the tissue sections with RNase or
by an excess of the complementary sense DNA during the hybridization
reaction. The histological staining of the tissue sections indicates
that tissue necrosis was negligible. Thus, a selective degeneration of
µ-receptor-expressing cells in the RVM by dermorphin-saporin
prevented abnormal pain resulting from SNL, without affecting normal
sensory responses.
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Figure 5.
Localization of opioid µ receptor mRNA in
frontal sections (20 µm) of the brainstem at the level of the caudal
raphe nuclei (approximately 11.0 mm from bregma) by a colorimetric
immunodetection method using a fluorescein-labeled, partial cDNA probe
generated from the coding region of the rat MOR. Hybridized
probe was detected by an anti-fluorescein antibody. Bright-field
micrographs (D-F) show the distribution of the
probe as a red stain (fast red). The sections were counterstained with
Mayer's hematoxylin (blue nuclei). Computer-assisted
mapping of representative sections shows the location of opioid µ receptor mRNA-labeled cells (dots) in the RVM of rats
that had been pretreated with dermorphin (A),
saporin (B), or dermorphin-saporin
(C) and the corresponding high-magnification,
bright-field micrographs (D-F) taken from the
same area in the raphe magnus (denoted by arrow in
A) of A-C, respectively. The cDNA probe
labeled discrete neuronal cell bodies. Two types of staining by fast
red are seen, (1) perinuclear staining (closed
arrowheads) and (2) cytoplasmic staining (open
arrowheads). Unlabeled cells can be seen as blue
nuclei with negligible or very light
blue cytoplasmic stain. In the RVM, cells that expressed opioid µ receptor transcripts were highly localized to the RM
and the MARN and also found at a lower cell density in
the PGRNl and the GRN. The localization
of the cells that express µ receptor mRNA is consistent with that
described previously. The RVM of dermorphin-saporin-pretreated rats
(C, F) consists of significantly fewer labeled
cells when compared with that of dermorphin-pretreated (A,
D) or saporin-pretreated (B, E) rats. The
specificity of the labeling for µ-opioid receptor transcripts is also
supported by the consistency in the distribution pattern of labeling
across multiple brainstem sections and sections from multiple animals.
This labeling could be abolished by RNase A pretreatment of the tissue
sections or by the presence of an unlabeled, corresponding sense DNA
during hybridization. GRN, Gigantocellular reticular
nucleus; MARN, magnocellular reticular nucleus;
PGRNl, paragigantocellular reticular nucleus-lateral
part; py, pyramidal tract; RM, raphe
magnus; RPA, raphe pallidus; VII, facial
nucleus. Scale bars, 50 µm.
|
|
| |
DISCUSSION |
Mechanistic interpretation of the neuropathic state has generally
focused on injury-induced changes in peripheral nerves and in the
spinal dorsal horn. Spinal sensitization is believed to occur as a
consequence of the increased firing of primary afferent fibers and has
been thought to be a key element in nerve injury-induced pain (Wall and
Gutnick, 1974; Devor, 1991, 1994; Kajander et al., 1992; Fields et al.,
1997). The time course of increased discharge from injured nerves does
not appear to correlate perfectly, however, with the sustained nature
of nerve injury-induced pain. Although nerve injury-induced pain is
sustained essentially unchanged for many weeks (Chaplan et al., 1994;
Bian et al., 1995), data from several groups show that the discharge
rate of injured afferents declines significantly over just a few days
after the injury (Han et al., 2000; Liu et al., 2000). Large myelinated
fibers are spontaneously active in the postinjury state, and uninjured
adjacent fibers also discharge tonically (Boucher et al., 2000; Li et
al., 2000; Wu et al., 2001). The known projections of these fibers to
the brainstem, along with the observations of plasticity at levels as
far rostral as the midbrain after injury to peripheral nerves (Kovelowski et al., 2000), point to a role of supraspinal sites in the
nerve injury-induced pain state. This concept is supported by various
lesion studies of ascending pathways (Houghton et al., 1999; Sun et
al., 2001) as well as by the emergence of evidence of the critical
importance of descending pain facilitation pathways in nerve
injury-induced pain. Specifically, blockade of nerve injury-induced
pain is seen after RVM microinjection of lidocaine (Pertovaara et al.,
1996; Kovelowski et al., 2000) as well as by lesions of the DLF
(Ossipov et al., 2000).
The results of the present study suggest that a specific population of
RVM neurons, namely, those expressing opioid µ receptors, is critical
in the behavioral expression of experimental neuropathic pain. These
RVM cells display characteristics consistent with those characterized
previously as facilitatory or pronociceptive. The electrophysiologic
characteristics of the neurons of this region have been well
characterized and strongly point to this region as a likely source of
facilitation of nociceptive input (Fields et al., 1983; Fields and
Heinricher, 1985; Fields, 1992). One neuronal class, labeled "ON"
cells because of a firing burst recorded just before activation of a
nocifensive response, is believed responsible for descending
facilitation of nociception via both local interactions within the RVM
and descending systems projecting to the spinal cord (Fields et al.,
1991; Heinricher et al., 1992; Heinricher and Roychowdhury, 1997).
Manipulations that increase nociceptive responsiveness, thus indicating
facilitation, also increase ON cell activity (Heinricher et al., 1989;
Kaplan and Fields, 1991; Morgan and Fields, 1994; Fields and Basbaum, 1999). Importantly, this class of neurons is hypothesized to represent the population of µ-opioid receptor-expressing cells. In agreement with this hypothesis, systemic or RVM morphine produces a
naloxone-sensitive depression in spontaneous and evoked firing rates of
identified ON cells (Heinricher et al., 1992), whereas the firing
characteristics of other RVM neurons are not affected by the opiate
(Heinricher et al., 1992). Analogous studies in vitro show
that RVM µ-opioid agonists directly hyperpolarize "secondary
cells" (Pan et al., 1990). Such findings suggest that ON cells are
likely to be the only µ-opioid receptor-expressing cells in the RVM
(Heinricher et al., 1992).
Our data show that conjugated dermorphin-saporin retains affinity and
efficacy at the opioid µ receptor, suggesting that this molecule is
suitable for targeting cells that express these receptors. Selective
loss of µ-opioid-containing neurons was demonstrated by a significant
decrease in the number of cells positively labeled for µ receptor
transcript in animals pretreated with dermorphin-saporin but not with
dermorphin or saporin. Rats treated with RVM dermorphin or saporin
demonstrated an equivalent presence of labeling for µ-opioid mRNA.
Neurons that were not in the vicinity of the site of
dermorphin-saporin injection were spared, however, suggesting that the
loss of µ-opioid receptor-expressing cells was not the result of a
nonselective cytotoxicity or because of trauma resulting from cannula
implantation or microinjection.
RVM microinjection of dermorphin-saporin, or of unconjugated saporin
or dermorphin, did not produce any significant changes in response
thresholds to normally non-noxious mechanical or noxious thermal
stimuli of the paw when evaluated over a 28 d time course. Because
a significant depletion of cells expressing µ-opioid receptor transcript was seen in animals injected with RVM dermorphin-saporin, it appears that µ-opioid receptor-expressing cells do not participate in the response to non-noxious or noxious sensory thresholds. Subsequent experimental nerve injury, however, showed that although rats pretreated with RVM dermorphin or saporin developed the expected increased sensitivity to normally non-noxious mechanical and noxious thermal stimuli, those animals pretreated with RVM dermorphin-saporin did not. These findings indicate that depletion of RVM cells expressing µ-opioid receptors, presumably cells that mediate descending
facilitation to the spinal dorsal horn, prevents the expected
neuropathic pain state. In the absence of electrophysiological
investigation, it is unknown whether such lesioned cells represent the
previously characterized population of RVM cells referred to as ON
cells. The specificity of dermorphin-saporin for RVM cells expressing µ-opioid receptors was confirmed by the use of -FNA, a selective and irreversible µ-opioid receptor antagonist (Ward et al., 1982; Jiang et al., 1990). RVM microinjection of -FNA was shown to antagonize the antinociceptive effects of a receptor-selective opioid
µ, but not selective or , agonist indicating that the dose
administered selectively blocked µ-opioid receptors; the selectivity
of the antagonist is supported by similar results in previous studies
(Tiberi et al., 1988; Melchiorri et al., 1991). Administration of RVM
dermorphin-saporin in rats pretreated with -FNA showed that the
expected prevention of nerve injury-induced pain was blocked; -FNA
pretreatment did not alter the development of nerve injury-induced pain
in groups pretreated with either dermorphin or saporin.
In addition to the observation of prevention of nerve injury-induced
pain by RVM pretreatment with dermorphin-saporin, our data also
demonstrate that the behavioral signs of experimental neuropathic pain
can be reversed by targeting µ-opioid receptor-expressing cells in
this region. Administration of dermorphin-saporin, but not of
dermorphin or saporin, showed a time-related return to normal levels of
sensitivity to non-noxious mechanical or to noxious thermal stimuli in
nerve-injured rats. The reversal of established experimental
neuropathic pain demonstrates the importance of descending facilitation
in sustaining pain. Together, these observations suggest a requirement
for RVM µ receptor-expressing cells for the expression and
maintenance of nerve injury-induced pain. The data indicate the
importance of such cells under pathological, but not normal
physiological, conditions because reactions to light touch or to acute
noxious stimuli are unaltered in uninjured or sham-operated rats. These
findings are consistent with previous observations indicating a role
for supraspinal mechanisms of neuropathic pain (Mansikka and
Pertovaara, 1997; Bian et al., 1998; Pertovaara, 1998; Kovelowski et
al., 2000; Ossipov et al., 2000; Sun et al., 2001) and are in agreement
with data resulting from experiments with more generalized and
reversible blockade of RVM activity such as lidocaine injection
(Pertovaara et al., 1996; Kovelowski et al., 2000). Finally, the data
of the present study are consistent with observations showing reversal
of nerve injury-induced pain by physical disruption of the DLF (Ossipov
et al., 2000).
The results presented here are consistent with the hypothesis that the
presence and activity of descending pain facilitation cells in the RVM
are required for the expression of experimental neuropathic pain.
Furthermore, these neurons are likely to be the µ-opioid
receptor-expressing cells of the RVM. Because dermorphin-saporin was
shown to be effective in reversing established experimental pain, the
µ-opioid-expressing neurons should therefore represent an appropriate
target for the development of strategies for the treatment of abnormal
pain states. It is clearly more important to be able to treat
established neuropathic pain, because one cannot anticipate its
development. Critically, targeting mechanisms of descending pain
facilitation offers a novel approach to the alleviation of chronic,
pathological pain that does not alter normal sensitivity to innocuous
or noxious sensations. These observations raise many important
questions including the nature of the tonic activation in the RVM that
drives descending facilitatory systems and the spinal circuitry by
which such projections facilitate the transmission of pain. Increased
understanding of underlying mechanisms that may drive abnormal pain
would be relevant to the formulation of novel treatment protocols.
| |
FOOTNOTES |
Received March 15, 2001; revised April 26, 2001; accepted May 1, 2001.
We thank Dr. Naomi Rance for access to the image-assisted mapping
equipment and software as well as for her critical evaluation of the
in situ hybridization data.
Correspondence should be addressed to Dr. Frank Porreca, Department of
Pharmacology, College of Medicine, University of Arizona Health
Sciences Center, Tucson, AZ 85724. E-mail: frankp{at}u.arizona.edu.
| |
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3121 - 3133.
[Abstract]
[Full Text]
[PDF]
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J. D. Carlson, J. J. Maire, M. E. Martenson, and M. M. Heinricher
Sensitization of Pain-Modulating Neurons in the Rostral Ventromedial Medulla after Peripheral Nerve Injury
J. Neurosci.,
November 28, 2007;
27(48):
13222 - 13231.
[Abstract]
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R. Benoliel, J. Epstein, E. Eliav, R. Jurevic, and S. Elad
Orofacial Pain in Cancer: Part I--Mechanisms
Journal of Dental Research,
June 1, 2007;
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491 - 505.
[Abstract]
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W. Kincaid, M. J. Neubert, M. Xu, C. J. Kim, and M. M. Heinricher
Role for Medullary Pain Facilitating Neurons in Secondary Thermal Hyperalgesia
J Neurophysiol,
January 1, 2006;
95(1):
33 - 41.
[Abstract]
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P. Mason
DECONSTRUCTING ENDOGENOUS PAIN MODULATIONS
J Neurophysiol,
September 1, 2005;
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1659 - 1663.
[Abstract]
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Y.-P. Chen, S.-R. Chen, and H.-L. Pan
Systemic Morphine Inhibits Dorsal Horn Projection Neurons through Spinal Cholinergic System Independent of Descending Pathways
J. Pharmacol. Exp. Ther.,
August 1, 2005;
314(2):
611 - 617.
[Abstract]
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I. D. Meng, J. P. Johansen, I. Harasawa, and H. L. Fields
Kappa Opioids Inhibit Physiologically Identified Medullary Pain Modulating Neurons and Reduce Morphine Antinociception
J Neurophysiol,
March 1, 2005;
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1138 - 1144.
[Abstract]
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[PDF]
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S. G. Khasabov, J. R. Ghilardi, P. W. Mantyh, and D. A. Simone
Spinal Neurons That Express NK-1 Receptors Modulate Descending Controls That Project Through the Dorsolateral Funiculus
J Neurophysiol,
February 1, 2005;
93(2):
998 - 1006.
[Abstract]
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M. M. Heinricher and M. J. Neubert
Neural Basis for the Hyperalgesic Action of Cholecystokinin in the Rostral Ventromedial Medulla
J Neurophysiol,
October 1, 2004;
92(4):
1982 - 1989.
[Abstract]
[Full Text]
[PDF]
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T. S. Brink and P. Mason
Role for Raphe Magnus Neuronal Responses in the Behavioral Reactions to Colorectal Distension
J Neurophysiol,
October 1, 2004;
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2302 - 2311.
[Abstract]
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L. R. Gardell, T. W. Vanderah, S. E. Gardell, R. Wang, M. H. Ossipov, J. Lai, and F. Porreca
Enhanced Evoked Excitatory Transmitter Release in Experimental Neuropathy Requires Descending Facilitation
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September 10, 2003;
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[Abstract]
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B. Bie, H. L. Fields, J. T. Williams, and Z. Z. Pan
Roles of {alpha}1- and {alpha}2-Adrenoceptors in the Nucleus Raphe Magnus in Opioid Analgesia and Opioid Abstinence-Induced Hyperalgesia
J. Neurosci.,
August 27, 2003;
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[Abstract]
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B. Bie and Z. Z. Pan
Presynaptic Mechanism for Anti-Analgesic and Anti-Hyperalgesic Actions of {kappa}-Opioid Receptors
J. Neurosci.,
August 13, 2003;
23(19):
7262 - 7268.
[Abstract]
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H. Foo and P. Mason
Discharge of Raphe Magnus ON and OFF Cells Is Predictive of the Motor Facilitation Evoked by Repeated Laser Stimulation
J. Neurosci.,
March 1, 2003;
23(5):
1933 - 1940.
[Abstract]
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M. S. Gold, D. Weinreich, C.-S. Kim, R. Wang, J. Treanor, F. Porreca, and J. Lai
Redistribution of NaV1.8 in Uninjured Axons Enables Neuropathic Pain
J. Neurosci.,
January 1, 2003;
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[Abstract]
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S. Marinelli, C. W. Vaughan, S. A. Schnell, M. W. Wessendorf, and M. J. Christie
Rostral Ventromedial Medulla Neurons That Project to the Spinal Cord Express Multiple Opioid Receptor Phenotypes
J. Neurosci.,
December 15, 2002;
22(24):
10847 - 10855.
[Abstract]
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M. Zhuo and G. F. Gebhart
Modulation of Noxious and Non-Noxious Spinal Mechanical Transmission From the Rostral Medial Medulla in the Rat
J Neurophysiol,
December 1, 2002;
88(6):
2928 - 2941.
[Abstract]
[Full Text]
[PDF]
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S. G. Khasabov, S. D. Rogers, J. R. Ghilardi, C. M. Peters, P. W. Mantyh, and D. A. Simone
Spinal Neurons that Possess the Substance P Receptor Are Required for the Development of Central Sensitization
J. Neurosci.,
October 15, 2002;
22(20):
9086 - 9098.
[Abstract]
[Full Text]
[PDF]
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S. E. Burgess, L. R. Gardell, M. H. Ossipov, T. P. Malan Jr, T. W. Vanderah, J. Lai, and F. Porreca
Time-Dependent Descending Facilitation from the Rostral Ventromedial Medulla Maintains, But Does Not Initiate, Neuropathic Pain
J. Neurosci.,
June 15, 2002;
22(12):
5129 - 5136.
[Abstract]
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[PDF]
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TOP
ABSTRACT
INTRODUCTION
