Receptor Subtype Mediating the Adrenergic Sensitivity of Pain Behavior and Ectopic Discharges in Neuropathic Lewis Rats

Doo Hyun Lee,1 Xianzeng Liu,1 Hyun Taek Kim,1 Kyungsoon Chung,1,2 and Jin Mo Chung1,2,3

 1Marine Biomedical Institute;  2Department of Anatomy and Neurosciences;  3Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1069


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -adrenergic receptor (alpha -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 alpha -AR blocker. We examined the effects of specific alpha 1- and alpha 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 alpha 1- and alpha 2-AR antagonist) or terazosin (TRZ, an alpha 1-AR antagonist) but not by various alpha 2-AR antagonists (idazoxan, rauwolscine, or yohimbine), suggesting that the alpha 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 alpha 1-ARs. These results suggest that the sympathetic dependency of neuropathic pain in the Lewis strain of the rat is mediated by the alpha 1 subtype of AR.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -adrenergic receptors (alpha -AR) because phentolamine (PTL), a mixed alpha 1- and alpha 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, alpha 1 or alpha 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, alpha -AR blockers, such as PTL, have not consistently proven to be effective in reducing neuropathic pain behaviors. The lack of a consistent effect of alpha -AR blockers makes it difficult to study the subtype of alpha -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 alpha 1- and alpha 2-AR antagonist on the Lewis neuropathic rat, we examined the subtypes of alpha -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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

alpha -AR ANTAGONISTS. The effects of systemic (intraperitoneal) injection of alpha -AR antagonists on mechanical hypersensitivity were tested at the 1-wk postoperative (PO) time point. Tested alpha -AR antagonists were PTL (a mixed alpha 1- and alpha 2-AR antagonist, from RBI), terazosin (TRZ, an alpha 1-AR antagonist), idazoxan HCl (IDZ, an alpha 2-AR antagonist, from Sigma), rauwolscine HCl (an alpha 2-AR antagonist, from RBI), and yohimbine HCl (an alpha 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 kg-1 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 Abeta , 2-14 m/s for Adelta , and <2 m/s for C fibers (Harper and Lawson 1985; Ritter and Mendell 1992; Waddell and Lawson 1990).

For analysis of the effects of alpha -AR antagonists and agonists, the number of spikes per minute was calculated, and the numbers were compared before and after a treatment.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of alpha -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 alpha -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 alpha 1- and alpha 2-AR antagonist) or TRZ (5 mg/kg, an alpha 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 alpha 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 alpha 1-AR-mediated mechanism.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Box plots showing the effects of alpha -adrenergic receptor (alpha -AR) blockers on mechanical sensitivity of the foot. Mechanical sensitivity was expressed as foot withdrawal thresholds determined by the up-down method with graded strengths of von Frey filaments. Four groups (n = 9 each) of rats were tested before neuropathic surgery (PRE), 1 week after the surgery (BASE), and at 3 time points (1, 4, and 24 h) after an intraperitoneal injection of 1 of the following 4 agents: saline (SAL, vehicle control), idazoxan (IDZ, 5 mg/kg, an alpha 2-AR antagonist), phentolamine (PTL, 5 mg/kg, a mixed alpha 1- and alpha 2-AR antagonist), and terazosin (TRZ, 5 mg/kg, an alpha 1-AR antagonist). Box plot code: horizontal bar within each box, the median value; the top and bottom borders of each box, 75 and 25th percentile values; and error bars of each box, 90 and 10th percentile values. *: values significantly different from those in the saline control group (P < 0.05 by the Kruskal-Wallis ANOVA on ranks followed by the Dunnett's multiple comparisons test).

Because IDZ had no effect, we tested two other alpha 2-AR antagonists to make certain of the ineffectiveness of alpha 2-AR antagonists. Nine neuropathic rats (1 wk PO) were prepared, and mechanical sensitivity was measured before and after intraperitoneal injection of alpha 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 alpha 2-ARs are not involved in the mechanical allodynia that develops in neuropathic Lewis rats.

Effects of alpha -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 alpha -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 alpha -AR antagonists are shown in Fig. 2. After a 10-min recording of the baseline ongoing activity, either saline or a specific alpha -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.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. Effects of alpha -AR blockers on ectopic discharges (in vivo preparation) in the neuropathic Lewis rat. Single unit activity was recorded from teased L5 dorsal root filaments between 7 and 14 days after tight ligation of the L5 and L6 spinal nerves. Records show examples of peristimulus time histograms compiled from 4 separate units. Various alpha -AR antagonists (or saline) were injected intraperitoneally at time 0, and the activities were followed for either 60 or 120 min. PTL and TRZ reduced the rate of ectopic discharges, but IDZ had no effect. Top, inset: segment of action potential recording made before saline injection.

The effects of various alpha -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.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. Box plots showing the effects of alpha -AR blockers on ectopic discharges recorded in an in vivo preparation. Single-unit activity was recorded from teased L5 dorsal root filaments between 7 and 14 days after tight ligation of the L5 and L6 spinal nerves. Before any drug injection, the number of ectopic discharges during a 10-min period is collected as baseline activity, and subsequent data in 10-min blocks are expressed as the percent of the change from the baseline. Box plot codes are the same as in Fig. 1. *, values significantly different from those in the saline control group (P < 0.05 by the Kruskal-Wallis ANOVA on ranks followed by the Dunnett's multiple comparisons test).

Because alpha -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 alpha -AR antagonists. All three alpha -AR antagonists (IDZ, PTL, and TRZ) produced a transient decrease in blood pressure lasting for ~20 min.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Effects of alpha -AR blockers on systemic blood pressure in the neuropathic Lewis rat. Arterial blood pressure was monitored during ectopic discharge recording sessions as shown in Fig. 2. Various alpha -AR antagonists (or saline) were injected intraperitoneally at time 0, and the recordings were followed for 60 min after the injection. Both alpha 1- (TRZ) and alpha 2-AR antagonists (IDZ) produced a similar pattern of blood pressure changes lasting ~20 min.

Effects of alpha -AR antagonists and agonists on ectopic discharges (in vitro study)

To confirm the effects of alpha -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 Abeta fibers (CV: 14-69.4 m/s), and 10 units (22.2%) were Adelta 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.


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Matrix chart summarizing the responses of 48 individually recorded units to exogenous application of EP

Because the ectopic discharges of many units responded to exogenously applied EP, we investigated the subtype of alpha -AR mediating the responses. We tested the effects of other alpha -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 alpha 1-AR agonist, 10 µM, from Sigma) first, and after washing out UK 14,304 (UK1; an alpha 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 alpha -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 alpha 1-AR agonist but not an alpha 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 alpha 1-AR. In fact, an alpha 2-AR agonist tends to inhibit ectopic discharges.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Effects of alpha -AR agonists on ectopic discharges recorded in an in vitro preparation. The L4 and L5 spinal nerves were tightly ligated in Lewis rats. Seven to 14 days later, single-unit activity was recorded from teased L4 or L5 dorsal root filaments with an in vitro recording setup, and alpha -AR agonists were applied to the perfusion bath. An example of responses to alpha -AR agonists is shown in A. Application of epinephrine (EP) evoked an enhancement of ectopic discharges, whereas UK 14,304 (UK1) produced a mild reduction. Application of phenylephrine (PEP) mimicked the effect of EP. Top, inset: segment of action potential recording made before EP injection. In B, responses to alpha -AR agonists on all tested units are shown as box plots. Activity was counted every minute, and data are expressed as percent changes from the baseline activity (before application of any agent). Box plot codes are the same as in Fig. 1. *, value significantly different from that in the EP group (P < 0.05 by the Kruskal-Wallis ANOVA on ranks followed by the Dunnett's multiple comparisons test).

We further investigated the subtype of alpha -AR mediating the EP evoked ectopic discharges by examining the effects of pretreatment with alpha -AR antagonists. Among the 29 units that showed an excitatory evoked response to EP, we tested the effects of alpha -AR antagonists on 11 units. We pretreated two units with IDZ, an alpha 2-AR antagonist, and two units with TRZ, an alpha 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).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6. Effects of alpha -AR antagonists on ectopic discharges recorded in an in vitro preparation. The L4 and L5 spinal nerves were tightly ligated in Lewis rats. Seven to 14 days later, single-unit activity was recorded from teased L4 or L5 dorsal root filaments with an in vitro recording setup and alpha -AR antagonists (along with EP) were applied to the perfusion bath. Examples of responses to alpha -AR antagonists are shown in A. Application of EP evoked an enhancement of ectopic discharges, and this enhancement was blocked by application of TRZ but not by IDZ. In B, responses to alpha -AR antagonists on all tested units are shown as box plots. Activity was counted every minute, and data are expressed as percent changes from the baseline activity (before application of any agent). Box plot codes are the same as in Fig. 1. *, value significantly different from that in the EP group (P < 0.05 by the Kruskal-Wallis ANOVA on ranks followed by the Dunnett's multiple comparisons test).

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 alpha 1- and alpha 2-AR ligand (EP) in the absence of alpha 1-AR (because of blockade by TRZ) produced an inhibition, presumably through activation of alpha 2-ARs.

The results indicate that the alpha 1-AR antagonist but not the alpha 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 alpha 1-AR. Furthermore, activation of alpha 2-AR seems to produce an inhibition of ectopic discharges.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha 1-AR (Davis et al. 1991). In fact, Stevens et al. (1993) reported that TRZ, an alpha 1-AR antagonist, effectively relieved SMP and vasospasm in a human patient. On the other hand, a number of animal studies suggests that alpha 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 alpha 1- and alpha 2-AR antagonist) (Lee et al. 1997). This study expands the previous work by showing that the subtype of alpha -AR mediating the reduction of mechanical hypersensitivity is alpha 1-AR, not alpha 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 alpha 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 alpha 2-AR antagonist (yohimbine or idazoxan) but not by an alpha 1-AR antagonist (prazosin) (Chen et al. 1996; Leem et al. 1997; Xie et al. 1995). From these observations, it was suggested that alpha 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 alpha 1-AR mediates neuropathic pain behaviors as well as ectopic discharges. However, it should be emphasized that the alpha 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 alpha -AR antagonists. Our data suggest that ongoing ectopic discharges are in part maintained by an alpha 1-AR-mediated mechanism.

It is also possible that different subtypes of alpha -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 alpha -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 alpha 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 alpha 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 alpha 2-AR is approximately five times higher than that of yohimbine (YHB) (Doxey et al. 1984). YHB is a nonimidazoline alpha 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 alpha -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 alpha -AR antagonists, whereas reductions of ectopic discharges do not begin until 30 min after the injection, and 2) both alpha 1- and alpha 2-AR antagonists influence blood pressure similarly, whereas a reduction of ectopic discharges occurs only after injection of alpha 1- but not after alpha 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 alpha 1-AR in modulation of ectopic discharges in in vitro experiments further suggests that the action of the alpha 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 alpha -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 alpha 1-AR agonist evokes an enhancement of ectopic discharges, whereas an alpha 2-AR agonist depresses them. An alpha 2-AR-mediated depression of ectopic discharges could also be seen when EP was applied after pretreatment with TRZ, an alpha 1-AR blocker. These excitatory and inhibitory actions of alpha 1- and alpha 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 alpha 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 alpha 1-AR-mediated mechanism. These results suggest that the potential contribution of alpha 1-AR in generation of neuropathic pain in human patients needs to be examined more carefully.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society