1Marine Biomedical Institute; 2Department of Anatomy and Neurosciences; 3Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Lee, Doo Hyun,
Xianzeng Liu,
Hyun Taek Kim,
Kyungsoon Chung, and
Jin Mo Chung.
Receptor subtype mediating the adrenergic sensitivity of pain behavior
and ectopic discharges in neuropathic Lewis rats. We attempted
to identify the subtype of -adrenergic receptor (
-AR) that is
responsible for the sympathetic (adrenergic) dependency of neuropathic
pain in the segmental spinal injury (SSI) model in the Lewis strain of
rat. This model was chosen because our previous study showed that pain
behaviors in this condition are particularly sensitive to systemic
injection of phentolamine (PTL), a general
-AR blocker. We examined
the effects of specific
1- and
2-AR
blockers on 1) behavioral signs of mechanical allodynia, 2) ectopic discharges recorded in the in vivo condition, and
3) ectopic discharges recorded in an in vitro setup. One
week after tight ligation of the L5 and L6 spinal nerves, mechanical
thresholds of the paw for foot withdrawals were drastically lowered; we
interpreted this change as a sign of mechanical allodynia. Signs of
mechanical allodynia were significantly relieved by a systemic
injection of PTL (a mixed
1- and
2-AR
antagonist) or terazosin (TRZ, an
1-AR antagonist) but
not by various
2-AR antagonists (idazoxan, rauwolscine,
or yohimbine), suggesting that the
1-AR is in part the
mediator of the signs of mechanical allodynia. Ongoing ectopic discharges were recorded from injured afferents in fascicles of the L5
dorsal root of the neuropathic rat with an in vivo recording setup.
Ongoing discharge rate was significantly reduced after intraperitoneal
injection of PTL or TRZ but not by idazoxan. In addition, by using an
in vitro recording setup, spontaneous activity was recorded from teased
dorsal root fibers in a segment in which the spinal nerve was
previously ligated. Application of epinephrine to the perfusion bath
enhanced ongoing discharges. This evoked activity was blocked by
pretreatment with TRZ but not with idazoxan. This study demonstrated
that both behavioral signs of mechanical allodynia and ectopic
discharges of injured afferents in the Lewis neuropathic rat are in
part mediated by mechanisms involving
1-ARs. These
results suggest that the sympathetic dependency of neuropathic pain in
the Lewis strain of the rat is mediated by the
1 subtype of AR.
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INTRODUCTION |
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It was well documented that neuropathic pain resulting
from peripheral nerve injury can be relieved in some patients by
blocking sympathetic outflow to the affected region (Bonica
1990; Loh and Nathan 1978
; Loh et al.
1980
). This type of neuropathic pain is referred to as
sympathetically maintained pain (SMP) and is contrasted to
sympathetically independent pain, which is not influenced by sympathetic manipulation (Roberts 1986
). At least a part
of this sympathetic dependency appears to be mediated by
-adrenergic receptors (
-AR) because phentolamine (PTL), a mixed
1- and
2-AR antagonist, was used
successfully as a diagnostic tool for identification of SMP patients
(Arnér 1991
; Raja et al. 1991
).
However, which subtype of AR is involved,
1 or
2, is highly controversial. It is obviously important to
identify the subtype to improve the means of treatment of SMP patients.
Sympathetic dependency of pain behaviors has also been shown in animal
models of neuropathic pain. Surgical or chemical sympathectomy has been
shown to be effective in relieving pain behaviors in various rat models
(Kim et al. 1997; Lee et al. 1997
;
Neil et al. 1991
; Shir and Seltzer 1991
).
However,
-AR blockers, such as PTL, have not consistently proven to
be effective in reducing neuropathic pain behaviors. The lack of a
consistent effect of
-AR blockers makes it difficult to study the
subtype of
-AR involved in neuropathic pain behaviors. In our recent
study (Lee et al. 1997
), we found a striking difference
in adrenergic sensitivity of neuropathic pain behaviors among different
strains of rats. Lewis rats in particular showed a powerful and
consistent antiallodynic response to systemically injected PTL. Because
of this robust effect of a mixed
1- and
2-AR antagonist on the Lewis neuropathic rat, we
examined the subtypes of
-AR that mediate both the adrenergic dependency of pain behaviors and the ectopic discharges of injured sensory neurons in this strain.
Preliminary data were presented in abstract form (Lee and Chung
1997; Lee et al. 1998
).
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METHODS |
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The experimental protocols were approved by the Animal Care and Use Committee of the University of Texas Medical Branch and were performed in accordance with the NIH guidelines.
Animals and surgery
Ninety-nine Lewis strain male rats (Harlan Sprague-Dawley,
Indianapolis, IN), weighing 150-250 g, were used in this study. The
animals were housed in groups of three, in plastic cages, with soft
bedding, under a 12/12-h reversed light-dark cycle (light cycle: 9:00
P.M. to 9:00 A.M.; dark
cycle: 9:00 A.M. to 9:00
P.M.). They were kept in the same room at a
constant ambient temperature and had free access to food and water. The
rats were kept 7 days under these conditions before experimental
manipulations. The study was performed during the dark cycle, which is
the rat's active period.
Neuropathic surgery was done as previously described in detail
(Choi et al. 1994; Kim and Chung 1992
).
Briefly, under gaseous anesthesia with a mixture of halothane (2% for
induction and 0.8% for maintenance) and a 2:1 flow of O2
and N2O, the left L5 and L6 spinal nerves were ligated
tightly with 6-0 silk threads. Wounds were closed and anesthesia was
discontinued. Animals were kept on a warm plate until they recovered
from anesthesia completely and resumed normal activities.
Behavioral tests
MECHANICAL ALLODYNIA.
Mechanical sensitivity of the paw was measured by determining the
median 50% foot withdrawal threshold with von Frey filaments with the
up-down method (Chaplan et al. 1994). The rats were
placed under a plastic cover (8 × 8 × 18 cm) on a metal
mesh floor, and von Frey filaments were applied from underneath to the
plantar surface of the foot. The area tested was the proximal one-half of the third or fourth toe. The toe was stimulated with a series of
eight von Frey filaments with logarithmically incremental bending forces (4.4, 7.4, 12.3, 21.6, 32.3, 53.9, 82.3, and 142.1 mN). The von
Frey filament was presented perpendicular to the toe surface with
sufficient force to bend it slightly and was held for ~2-3 s. An
abrupt withdrawal of the foot (hind limb flinching) during stimulation
or immediately after the removal of stimulus was considered to be a
positive response. On the basis of electrophysiological recordings, we
found that thresholds for mechanoreceptors in the rat foot did not
exceed 25 mN, whereas those of nociceptors were rarely lower than that
level (Leem et al. 1993
). However, the threshold for
hind limb flinching after neuropathic surgery often became <10 mN,
suggesting that a nociceptive reflex was elicited by the activation of
mechanoreceptors in this situation. Therefore a significant reduction
in the mechanical threshold for hind limb flinching was interpreted as
a sign of mechanical allodynia.
-AR ANTAGONISTS.
The effects of systemic (intraperitoneal) injection of
-AR
antagonists on mechanical hypersensitivity were tested at the 1-wk
postoperative (PO) time point. Tested
-AR antagonists were PTL (a
mixed
1- and
2-AR antagonist, from RBI),
terazosin (TRZ, an
1-AR antagonist), idazoxan HCl (IDZ,
an
2-AR antagonist, from Sigma), rauwolscine HCl (an
2-AR antagonist, from RBI), and yohimbine HCl (an
2-AR antagonist, from Sigma).
Electrophysiological studies
IN VIVO STUDY.
Single-unit recordings were made from filaments of the left L5 dorsal
root in neuropathic rats at a time between 7 and14 PO days. The rats
were anesthetized with a mixture of halothane and a 2:1 ratio of
O2 and N2O. The left jugular vein was
cannulated with a polyethylene tube (PE-20) for systemic drug
administration. The right carotid artery was cannulated to monitor
blood pressure throughout the experiment. When the diastolic blood
pressure dropped to <60 mmHg for >30 min, the experiment was
discontinued. Under artificial ventilation, animals were paralyzed with
pancuronium bromide (Parvlon: a single bolus of 1 mg/kg iv followed by
a continuous intravenous infusion, 0.4 mg kg1
h
1). The ventilator was adjusted to an end-tidal
CO2 level between 4 and 5% throughout the experiment. The
spinal cord was exposed by a laminectomy of the L1-L6 vertebrae. The
animal was mounted on a spinal investigation frame, and a heated
mineral oil pool (36°C) was made over the exposed tissue to prevent
it from drying. The L5 dorsal root was cut near the spinal cord, and
the distal stump was placed on a mirror plate. Fine filaments were
dissected until a single spontaneous unit could be isolated on the
basis of its amplitude and waveform. The unit activity was amplified with an AC-coupled amplifier (WPI, DAM-5A) and led to a window discriminator (Mentor, N-750). The output of the window discriminator was used to compile peristimulus time histograms by a data acquisition system (CED-1401, Spike 2).
IN VITRO STUDY.
For the in vitro study, the left L4 and L5 spinal nerves were ligated.
Seven to 14 days later, animals were anesthetized with halothane, and
the L4 and L5 dorsal root ganglia (DRG), along with dorsal roots and
spinal nerves, were removed. The DRG were placed in an in vitro
recording chamber that consisted of two separate compartments, one for
the dorsal root and the other for the DRG and spinal nerve. The DRG and
spinal nerve compartment was perfused with oxygenated (95%
O2-5% CO2) artificial cerebrospinal fluid
[composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 10 dextrose,
1.2 MgCl2, 1.2 CaCl2, pH 7.3] at a rate of 4-5 ml/min. The dorsal root compartment was filled with mineral oil.
The temperature was kept at 35 ± 1°C by means of a
temperature-controlled water bath. Ectopic discharges were recorded
from the teased dorsal root fascicles, and the spinal nerve was
stimulated with a suction electrode. Fiber types were classified
according to their conduction velocity: >14 m/s for A, 2-14 m/s
for A
, and <2 m/s for C fibers (Harper and Lawson
1985
; Ritter and Mendell 1992
; Waddell
and Lawson 1990
).
Statistical treatments
Data are displayed as box plots, and differences between groups were tested with the Kruskal-Wallis one-way analysis of variance followed by the Dunnett's post hoc multiple comparisons test. Two-tailed P values <0.05 were considered to be significant.
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RESULTS |
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Effects of -AR antagonists on mechanical allodynia
Nine rats were used to examine the time course of neuropathic pain
behaviors in Lewis strain rats after segmental spinal nerve injury
(SSI). All rats showed mechanical allodynia (decreased hind limb
flinching threshold) that reached the peak level 1 day after nerve
injury. This high level of mechanical sensitivity was maintained for
the next 8 wk. Although the mechanical threshold gradually increased
beyond 8 wk, a significant level of hypersensitivity was maintained for
the entire observation period of 20 wk.
The effects of -AR antagonists on mechanical hypersensitivity were
examined on 36 (9 in each group) neuropathic rats, and the results are
shown in Fig. 1. Neuropathic surgery
produced mechanical hypersensitivity at 1 wk PO, and so the hind limb
flinching threshold was reduced to a very low level (BASE in Fig. 1).
Intraperitoneal injection of PTL (5 mg/kg, a mixed
1-
and
2-AR antagonist) or TRZ (5 mg/kg, an
1-AR antagonist) produced a significant elevation of the
threshold for 1-4 h. On the other hand, neither saline nor IDZ (5 mg/kg, an
2-AR antagonist) had any effect on mechanical hypersensitivity. These data suggest that the mechanical allodynic behavior of neuropathic Lewis rats is in part maintained by an
1-AR-mediated mechanism.
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Because IDZ had no effect, we tested two other 2-AR
antagonists to make certain of the ineffectiveness of
2-AR antagonists. Nine neuropathic rats (1 wk PO) were
prepared, and mechanical sensitivity was measured before and after
intraperitoneal injection of
2-AR antagonists. On a
given day, one of the following three substances, rauwolscine (5 mg/kg), yohimbine (5 mg/kg), or saline (0.2 ml), was administered to
each of three groups of randomly selected rats. The procedure was
repeated a total of three times on 3 consecutive days according to the
Latin Square design so that each rat received all three substances in
random order over 3 days. None of these compounds had any effect on the
mechanical thresholds for foot withdrawal. Therefore it seems clear
that
2-ARs are not involved in the mechanical allodynia
that develops in neuropathic Lewis rats.
Effects of -AR antagonists on ectopic discharges (in vivo study)
Because ectopic discharges of injured afferents are an important
underlying mechanism of neuropathic pain behaviors, we examined the
effects of -AR antagonists on the ectopic discharges of 44 units (10 units for saline, 13 for PTL, 12 for TRZ, and 9 for IDZ) recorded from
the L5 dorsal roots of 25 Lewis rats between 7 and 14 days after tight
ligation of the L5 and L6 spinal nerves. Many afferent fibers in the L5
dorsal root of neuropathic rats showed ongoing activity without any
apparent stimulation. Because the L5 spinal nerve was tightly ligated
at the time of neuropathic surgery, all afferent fibers in the L5
dorsal root were disconnected from their original sensory receptors,
where normal impulse generation occurs. The recorded ongoing activity
therefore must originate from sites other than the original receptors;
thus they are regarded as ectopic discharges. Initially, the firing
rate of each ectopic discharge was recorded for
10 min, and this rate
was considered the baseline ectopic discharge rate. Then a bolus of PTL
(2.5 mg/kg), TRZ (2.5 mg/kg), IDZ (2.5 mg/kg), or saline (0.2 ml) was injected intraperitoneally. The recording was maintained for
1 h
after the administration of each drug and extended to 2 h in some cases.
Examples of single-unit recordings of ectopic discharges and the
effects of -AR antagonists are shown in Fig.
2. After a 10-min recording of the
baseline ongoing activity, either saline or a specific
-AR
antagonist was injected. Both PTL and TRZ reduced the rate of ectopic
discharges with a delay of 10-20 min after the injection. The rate
then recovered in 1.5-2 h. Neither saline nor IDZ had any effect.
Testing with intravenous injection (1 mg/kg) of PTL in two other units
(recorded from two animals) produced a similar reduction in the ectopic
discharges.
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The effects of various -AR antagonists examined in all 44 units were
analyzed. As shown in Fig. 3, PTL and TRZ
significantly reduced the rate of ectopic discharges, whereas neither
saline nor IDZ produced any effect.
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Because -AR antagonists have strong cardiovascular effects, it was
necessary to monitor blood pressure very closely during ectopic
discharge recordings. Figure 4 shows
examples of blood pressure responses to injections of various
-AR
antagonists. All three
-AR antagonists (IDZ, PTL, and TRZ) produced
a transient decrease in blood pressure lasting for ~20 min.
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Effects of -AR antagonists and agonists on ectopic discharges
(in vitro study)
To confirm the effects of -AR antagonists in simplified
conditions, we tested them on ectopic discharges recorded in an in vitro preparation. Single-unit recordings were made from teased L4 or
L5 dorsal root filaments between 7 and 14 days after tight ligation of
the L4 and L5 spinal nerves. Table 1
summarizes the general characteristics of the 48 recorded units (from
20 rats) and responsiveness to exogenously applied epinephrine
bitartrate (EP, from RBI). The conduction velocity was measured for 45 of 48 units. Thirty-five of 45 units (77.8%) were A
fibers (CV: 14-69.4 m/s), and 10 units (22.2%) were A
fibers (CV: 7.8-13 m/s). We did not find any C fibers in this study. Application of EP to
the perfusion bath evoked an enhancement of the discharges by >30%
over the baseline in 29 units (60.4%), whereas 16 units (33.3%) did
not show any response. A small number of units (4 units, 6.3%) showed
a decreased (reduction of >30% of the baseline value) ectopic
discharge rate in response to EP.
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Because the ectopic discharges of many units responded to exogenously
applied EP, we investigated the subtype of -AR mediating the
responses. We tested the effects of other
-AR agonists on 7 units
among the 29 units that showed an excitatory evoked response to EP.
Among the seven units tested, three units received
L-phenylephrine HCl (PEP; an
1-AR agonist,
10 µM, from Sigma) first, and after washing out UK 14,304 (UK1; an
2-AR agonist, 10 µM, from RBI) was applied. The order
of application of PEP and UK1 was reversed in the remaining four units.
Figure 5A shows an example of
responses to
-AR agonists, and the results of all the units are
summarized in Fig. 5B. Infusion of EP produced an
enhancement of ectopic discharges of 104.3% (median value) over the
baseline rate. Application of PEP produced a similar enhancement of
discharges (median value of 45.5% over the baseline). On the other
hand, UK1 failed to induce an enhancement of ectopic discharges (median
value of 38.2% reduction from the baseline). The results indicate that
an
1-AR agonist but not an
2-AR agonist
can mimic the action of EP on ectopic discharges and suggest that the
ectopic discharges evoked by EP in axotomized sensory neurons are
mediated by
1-AR. In fact, an
2-AR
agonist tends to inhibit ectopic discharges.
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We further investigated the subtype of -AR mediating the EP evoked
ectopic discharges by examining the effects of pretreatment with
-AR
antagonists. Among the 29 units that showed an excitatory evoked
response to EP, we tested the effects of
-AR antagonists on 11 units. We pretreated two units with IDZ, an
2-AR
antagonist, and two units with TRZ, an
1-AR antagonist,
before application of EP. In both cases, pretreatment with IDZ did not
influence the EP-evoked ectopic discharges, whereas pretreatment with
TRZ produced a long-lasting blockade of the EP-induced enhancement of
ectopic discharges. We tested the remaining seven units for the effect
of pretreatment with IDZ first and then, after washing out the drug,
the effect of TRZ. Figure 6A
shows an example of this experiment, and the results of all units are
summarized in Fig. 6B. Infusion of EP alone on these units
produced an enhancement of ectopic discharges of 95.8% (median value)
over the baseline rate. After pretreatment with IDZ, the application of
EP still produced a similar enhancement of discharges (median value of 53.8% increase over the baseline). On the other hand, pretreatment with TRZ completely blocked EP-evoked enhancement of ectopic discharges (median value of 44.7% reduction from the baseline).
|
Pretreatment with TRZ produced long-lasting effects. On six units,
reapplication of EP 30-60 min after pretreatment with TRZ evoked an
inhibition (median value of 35.9% reduction from the baseline) rather
than excitation. Thus application of a mixed 1- and
2-AR ligand (EP) in the absence of
1-AR
(because of blockade by TRZ) produced an inhibition, presumably through
activation of
2-ARs.
The results indicate that the 1-AR antagonist but not
the
2-AR antagonist blocks the action of EP on ectopic
discharges and again suggest that the ectopic discharges evoked by EP
in axotomized sensory neurons are mediated by
1-AR.
Furthermore, activation of
2-AR seems to produce an
inhibition of ectopic discharges.
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DISCUSSION |
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The role of the sympathetic nervous system in neuropathic pain is
a complex and controversial issue. In particular, although it is
generally accepted that the -AR is involved in SMP patients, it is
not clear which subtype is playing the important role. One line of
evidence, mainly obtained from human patients, supports the importance
of
1-AR (Davis et al. 1991
). In fact,
Stevens et al. (1993)
reported that TRZ, an
1-AR
antagonist, effectively relieved SMP and vasospasm in a human patient.
On the other hand, a number of animal studies suggests that
2-AR is more important (Chen et al. 1996
;
Leem et al. 1997
; Sato and Perl 1991
;
Xie et al. 1995
). In our previous study, we reported
that the mechanical hypersensitivity that develops in the SSI model in
the Lewis strain of rats was greatly reduced by intraperitoneal
injection of PTL (a mixed
1- and
2-AR
antagonist) (Lee et al. 1997
). This study expands the
previous work by showing that the subtype of
-AR mediating the
reduction of mechanical hypersensitivity is
1-AR, not
2-AR. Furthermore, both in vivo and in vitro
physiological experiments showed that adrenergic sensitivity of ectopic
discharges in an axotomized sensory neuron is mediated by
1-AR.
It is well known that sensory neurons develop abnormal, spontaneous
activity after being separated from their peripheral receptors by
axotomy (Devor and Jänig 1981; Devor et al.
1994
; Kajander and Bennett 1992
; Korenman
and Devor 1981
; Scadding 1981
; Wall and
Gutnick 1974
). This abnormal, spontaneous activity is due to
ectopically generated discharges and is considered to be an important
contributor to central sensitization (Woolf 1995
), and thus the activity plays a major role in the development and maintenance of neuropathic pain (Gracely et al. 1992
; Sheen
and Chung 1993
; Yoon et al. 1996
). Although the
precise generation mechanisms and factors influencing ectopic
discharges are not clear yet, sympathetic manipulations are known to
influence the rate of the discharges. It was reported that sympathetic
stimulation (Devor et al. 1994
; McLachlan et al.
1993
) or application of norepinephrine (Chen et al.
1996
; Devor et al. 1994
; Wall and Gutnick
1974
; Xie et al. 1995
) increases the rate of
ectopic discharges. These sympathetically evoked ectopic discharges are
usually blocked by an
2-AR antagonist (yohimbine or
idazoxan) but not by an
1-AR antagonist (prazosin) (Chen et al. 1996
; Leem et al. 1997
;
Xie et al. 1995
). From these observations, it was
suggested that
2-AR is responsible for mediating the
adrenergic dependency of ectopic discharges. The results of these
studies appear to contradict those of the current study because our
data show that the
1-AR mediates neuropathic pain behaviors as well as ectopic discharges. However, it should be emphasized that the
2-AR-mediated responses reported by
all of these previous studies are for sympathetically evoked
discharges, not for ongoing discharges. In fact, until the current
study, ongoing discharges have never been shown to be affected by any
-AR antagonists. Our data suggest that ongoing ectopic discharges are in part maintained by an
1-AR-mediated mechanism.
It is also possible that different subtypes of -AR are involved in
mediating adrenergic sensitivity of pain behaviors in different strains
of animals. The current study used the Lewis strain of rats, whereas
the studies by Devor et al. (1994)
and Chen et al. (1996)
used the
Wistar-derived Sabra strain of rats, and the one by Xie et al. (1995)
used the Sprague-Dawley strain. Considering that the Lewis strain of
rats is known to release a smaller quantity of norepinephrine in a
stress condition than other strains (Dhabhar et al.
1993
; Sternberg et al. 1992
), it is possible
that the Lewis strain represents a unique population of rats. The
higher degree of adrenergic dependency of pain behaviors in Lewis rats
compared with other strains (Lee et al. 1997
) also suggests that strain difference may be an important factor in determining the degree of adrenergic dependency of pain behaviors as
well as the subtype of
-AR mediating the effect.
Another complicating factor for comparing different studies is
potential nonspecificity of various adrenergic agents used by each
study. TRZ is a close structural analogue of the well-known 1-AR antagonist prazosin. It is more soluble in water
than prazosin, and it has a longer half-life (Hoffman and
Lefkowitz 1996
). UK 14,304 is known to have higher specificity
for the
2-AR than clonidine (Andorn et al.
1988
; Paris et al. 1989
). IDZ has an imidazoline
structure and interacts with both I1 and I2
imidazoline receptors (Langin et al. 1990
), but the
specificity of IDZ for
2-AR is approximately five times
higher than that of yohimbine (YHB) (Doxey et al. 1984
).
YHB is a nonimidazoline
2-AR antagonist, but it is known
to block sodium channels in the giant squid axon (Lipicky et al.
1978
) as well as in the mouse brain (Huang et al.
1978
; Zimanyi et al. 1988
) in a use-dependent
manner. In this study, neuropathic pain behavior was not reduced by
either IDZ or YHB. For physiological study, we used IDZ exclusively
because the voltage-gated sodium channels were suggested to be an
important source of ectopic discharge generation, and YHB may interfere with these channels. Although IDZ may interact with imidazoline receptors, this seems to be more likely a problem in the CNS
(King et al. 1995
).
Because -AR antagonists have strong cardiovascular effects, it is
possible that the changes in the rate of ectopic discharges in in vivo
experiments are secondary to the changes in blood pressure of the
animal. However, monitoring systemic blood pressure while recording
ectopic discharges revealed that 1) most blood pressure changes occur during the first 20 min after injection of
-AR antagonists, whereas reductions of ectopic discharges do not begin until 30 min after the injection, and 2) both
1- and
2-AR antagonists influence blood
pressure similarly, whereas a reduction of ectopic discharges occurs
only after injection of
1- but not after
2-AR antagonist injection. These mismatches between
changes in blood pressure and ectopic discharges suggest that the
former is not a direct cause of the latter. In addition, the
specificity of
1-AR in modulation of ectopic discharges
in in vitro experiments further suggests that the action of the
1-AR antagonist is not a side effect.
Our approach of recording ectopic discharges may appear to be
inconsistent because we focused on spontaneous activity in the in vivo
study on one hand and on evoked activity in the in vitro study on the
other hand. Because our goal is to find out the subtype of -AR that
is involved in SMP, it is essential to study "sympathetically maintained ectopic discharges." Sympathetically maintained ectopic discharges are the activity that is present in the resting state, and
hence these are influenced only by the release of adrenergic compounds
because of basal sympathetic tone and circulating catecholamines. Therefore we examined spontaneous ectopic discharges in the in vivo
experiments. The experimental conditions of the in vitro study,
however, were different from that of the in vivo study. During
isolation of the tissue for recording, the sympathetic supply is
invariably denervated, and hence there is no longer basal sympathetic
tone. Because we did not add catecholamines to the perfusion solution,
there were no agents comparable with circulating catecholamines either.
Therefore, when we evoke activity by adding adrenergic agonists to the
perfusion solution in the in vitro study, we presumably mimic the
presence of basal sympathetic tone and circulating catecholamines found
in the in vivo condition. Therefore we focused on evoked responses in
the in vitro study.
The results of in vitro experiments showed that an 1-AR
agonist evokes an enhancement of ectopic discharges, whereas an
2-AR agonist depresses them. An
2-AR-mediated depression of ectopic discharges could
also be seen when EP was applied after pretreatment with TRZ, an
1-AR blocker. These excitatory and inhibitory actions of
1- and
2-AR agonists are in agreement
with their general actions in the CNS (Millan et al.
1994
; Pieribone et al. 1994
) and in the
sympathetic ganglia (Akasu et al. 1985
; Brown and
Caulfield 1979
).
In conclusion, this study showed that the 1-AR in part
mediates neuropathic pain behaviors in Lewis strain rats. Furthermore, both in vivo and in vitro electrophysiological experiments showed that
ectopic discharges generated from injured afferents are also in part
dependent on an
1-AR-mediated mechanism. These results suggest that the potential contribution of
1-AR in
generation of neuropathic pain in human patients needs to be examined
more carefully.
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ACKNOWLEDGMENTS |
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We thank Drs. J. Zhang and R. H. LaMotte for showing us their in vitro electrophysiological setup. We also thank Abbott Laboratories for a generous gift of TRZ.
This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-31680, NS-35057, and NS-11255.
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FOOTNOTES |
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Address for reprint requests: J. M. Chung, Marine Biomedical Institute, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 September 1998; accepted in final form 26 January 1999.
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REFERENCES |
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