Department of Anesthesiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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Zhang, Jun-Ming, Huiqing Li, and Sorin J. Brull. Perfusion of the Mechanically Compressed Lumbar Ganglion With Lidocaine Reduces Mechanical Hyperalgesia and Allodynia in the Rat. J. Neurophysiol. 84: 798-805, 2000. The rat L5 dorsal root ganglion (DRG) was chronically compressed by inserting a hollow perforated rod into the intervertebral foramen. The DRG was constantly perfused through the hollow rod with either lidocaine or normal saline delivered by a subcutaneous osmotic pump. Behavioral evidence for neuropathic pain after DRG compression involved measuring the incidence of hindlimb withdrawals to both punctate indentations of the hind paw with mechanical probes exerting different bending forces (hyperalgesia) and to light stroking of the hind paw with a cotton wisp (tactile allodynia). Behavioral results showed that for saline-treated control rats: the withdrawal thresholds for the ipsilateral and contralateral paws to mechanical stimuli decreased significantly after surgery and the incidence of foot withdrawal to light stroking significantly increased on both ipsilateral and contralateral hind paws. Local perfusion of the compressed DRG with 2% lidocaine for 7 days at a low flow-rate (1 µl/h), or for 1 day at a high flow-rate (8 µl/h) partially reduced the decrease in the withdrawal thresholds on the ipsilateral foot but did not affect the contralateral foot. The incidence of foot withdrawal in response to light stroking with a cotton wisp decreased significantly on the ipsilateral foot and was completely abolished on the contralateral foot in the lidocaine treatment groups. This study demonstrated that compression of the L5 DRG induced a central pain syndrome that included bilateral mechanical hyperalgesia and tactile allodynia. Results also suggest that a lidocaine block, or a reduction in abnormal activity from the compressed ganglia to the spinal cord, could partially reduce mechanical hyperalgesia and tactile allodynia.
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INTRODUCTION |
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Animal models of neuropathic
pain are supported with behavioral evidence of spontaneous pain and
cutaneous hyperalgesia after injury of peripheral nerves
(Bennett and Xie 1988; Kim and Chung 1992
; Seltzer et al. 1990
) or dorsal root
ganglia (DRG) (Hu and Xing 1998
; Olmarker and
Myers 1998
; Song et al. 1999
). Although the
mechanism of neuropathic pain following nerve/ganglion injury is
unresolved, evidence suggests that somata of the DRG may become an
important source of pain after an injury of the peripheral nerve
(Devor and Obermayer 1984
). When rat peripheral nerves
or primary sensory neurons are injured, certain DRG neurons become hyperexcitable and may exhibit various patterns of abnormal ectopic discharge (Babbedge et al. 1996
; Burchiel
1984
; Hu and Xing 1998
; Kajander et al.
1992
; Wall and Devor 1983
; Xie et al.
1995
; Zhang et al. 1997b
, 1999
). The
ionic and cellular mechanisms of the increased excitability are not
known, but accumulation of certain types of sodium channels at the
injured site or the related DRGs may account for the changes in
membrane properties of the DRG somata and thus contribute to the
enhancement of neuronal excitability (Devor et al. 1993
;
Rizzo et al. 1995
; Zhang et al. 1997a
).
Evidence that modulation of sodium currents in the DRG may alter
neuropathic pain comes from neuropathic animal models where systemic
administration of lidocaine suppressed the ectopic discharges recorded
extracellularly in the neuroma, the DRG, or the spinal horn neurons
(Chabal et al. 1989; Devor et al. 1992
;
Omana-Zapata et al. 1997b
; Sotgiu et al.
1992
). Systemic lidocaine treatment also prevented the
development of thermal hyperalgesia and cutaneous thermal abnormalities
after peripheral nerve injury in rats (Sotgiu et al.
1995
). It is not known, however, to what extent the alteration in neuronal excitability and abnormal activities originating in the DRG
contribute to the development of pain and hyperalgesia.
In the present study, we investigated the neurological mechanisms of
cutaneous hyperalgesia and tactile allodynia using an animal model of
lumbar radiculopathy. In this model, a stainless steel, hollow,
perforated rod was inserted surgically into the intervertebral foramen
to produce a chronic compression of the L5 DRG.
The procedure was similar to the model described elsewhere (Hu
and Xing 1998; Song et al. 1999
) except that the
inserted solid rod used previously was replaced by a hollow rod
connected to an osmotic pump and that only one, not two DRGs, was
chronically compressed. The justification for this new animal model is
that clinically, single-ganglion compression/injury is more common than
two-ganglion-compression injuries. The purposes of this study were to
evaluate whether mechanical hyperalgesia and allodynia were present
after a chronic compression of the L5 DRG and to measure the changes in cutaneous sensitivity after blocking/reducing the input of abnormal activity from the compressed ganglion to the
spinal horn by delivering different rates of lidocaine to the
compressed ganglion in vivo.
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METHODS |
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Fifty-seven adult, male, Sprague-Dawley rats were used for all the experiments. At the start of experiment the animal weights were between 150 and 200 g. Animals were housed in groups (3 per cage) and were kept 3-4 days under these conditions before surgery, and up to 4 wk after surgery. The experimental protocol was approved by the University of Arkansas Medical School Animal Care and Use Committee.
Surgical procedure for implantation of hollow stainless steel rod and osmotic minipump
Rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg). With the rats in a prone position, an incision was made along the midline of the back at the L4 and L6 spinal level. Following separation of the right paraspinal muscles from the transverse processes, the L5 intervertebral foramen was exposed. An L-shaped rod made of hollow stainless steel (4 × 2 mm in length and 0.7 mm in diameter; Fig. 1) was carefully inserted into the foramen without performing a laminectomy. The rod was connected to an osmotic minipump (ALZET, Alza) via a piece of fine silicon tubing (0.51 mm ID, 0.94 OD, length: 60 mm, volume: 20 µl) (Dow Corning, Medical Materials Division). A surgical ligature was placed tightly around the silicon tubing (5 mm away from the rod), and the other end of the tubing was sutured to the spinal ligament to stabilize the inserted rod and prevent its migration. The minipump was implanted subcutaneously and secured by suturing to the spinal ligament. The implanted pumps offered an advantageous alternative to repeated injections. During drug delivery, no external connection was required, and rats were untethered and unrestrained, thereby minimizing animal handling stress. The incision was closed in layers and prophylactic amoxicillin/clavulanate potassium (Augmentin, SmithKline Beecham) was given to the rats daily in the drinking water (7.52 g in 500 ml) for at least three postoperative days.
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Perfusion of the compressed DRG
Implanted pumps had a volume of 200 µl, capable of delivering
solutions either at a rate of 1 µl/h for seven consecutive days or at
a rate of 8 µl/h for 24 h The implanted pump was filled with 2%
lidocaine or normal saline according to the methods described elsewhere
(Munson et al. 1997; Oyelese et al. 1997
;
Verge et al. 1995
). The tube leading from the pump was
filled with 10 µl of a Heparin solution (in 2% lidocaine or normal
saline) followed by 5 µl of Evan's Blue solution (1% Evan's Blue
in 2% lidocaine or normal saline). The osmotic pumps implanted in the
rat were preloaded with lidocaine or saline by an investigator other
than the one performing behavioral-testing to avoid possible
introduction of testing bias. At the end of each experiment, the
ganglion was carefully examined under a dissecting microscope. The
appearance of the dye in or around the ganglion was considered an
indication that the delivered drug reached the compressed ganglion.
Behavioral testing procedures
Animals were inspected and tested every day for 3 days prior to
surgery, every other day for the first two postoperative weeks, twice
during the 3rd week, and one last time at the end of 4 wk postoperatively for a total of 13 testing sessions. The testing procedure was described previously (Song et al. 1999).
Briefly, the rat was acclimatized, prior to the test, for 15 min and
tested in a closed Plexiglass box with a mesh floor (with 1 × 1 cm openings) through which mechanical stimuli were applied.
WITHDRAWAL TO PUNCTATE MECHANICAL STIMULATION OF THE FOOT. Mechanical probes consisting of nylon filaments that had bending forces of 5, 10, 20, 40, 60, 80, 100, 120, and 160 mN, and 100-µm cylindrical tips were applied to the plantar surface of the foot from underneath the cage floor. Each filament was applied once to 10 different predetermined locations on the ventral surface, spaced across nearly the entire extent of the paw. The duration of each stimulus was 1 s and the interstimulus interval was 10-15 s. The mechanical probes were applied in the order of increasing bending force, with a given filament delivered to each spot alternatively from one paw to the other in sequence (from the first to the tenth spot) until a 100% response to certain force was evoked on both paws.
The modified filaments differed in some respects from the conventional von Frey filaments used to measure withdrawal thresholds to mechanical stimulation of the rat hind paw. The bending force of the modified filaments was independent of the filament diameter. The diameter of the tip was held constant by attaching 100-µm-diam rods to nylon filaments of differing diameter to deliver a "blunt pinprick" to the ventral surface of the hind paw. The advantage of this method was that preoperative thresholds were easily obtained without the need for thicker filaments with blunt tip diameters that could sometimes lift the paw under pressure without eliciting a withdrawal response.CALCULATION OF THE WITHDRAWAL THRESHOLDS TO
MECHANICAL STIMULATION.
Since the percent withdrawal responses plotted as a function of the
stimulus magnitudes (bending forces) were hyperbolic, but not linear,
the Hill equation, P = Pmax
S/(S50
+ S
), which provided the best
nonlinear least square curve-fit to the data, was used to estimate the
S50 and the slope factor (
). The
parameters in the equation represent percent response (P), the maximal response or 100% withdrawal response
(Pmax), the stimulus magnitude
(S), the stimulus magnitude or the force associated with
50% response of foot withdrawal
(S50), and a slope factor (
), known
as the Hill coefficient. Fitting the data to the Hill equation was done
using a curve-fitting program (Microcal Origin 5.0, Microcal Software,
Northampton, MA).
FOOT WITHDRAWAL TO INNOCUOUS MECHANICAL STIMULI. A wisp of cotton pulled up but still attached to a cotton swab was stroked mediolaterally across the plantar surface of the skin through the floor of the plastic cage. Six strokes were delivered to each foot, alternating between right and left with each stroke. The duration of each stroke was 1 s, and the inter-stroking interval was 10-15 s. A single, quick withdrawal reflex was considered to indicate the presence of tactile allodynia.
Statistical analysis
Data were expressed as means and standard errors of the mean (SE). Differences in withdrawal thresholds over time were tested using Friedman repeated-measures ANOVA on Ranks followed by post hoc pairwise comparisons. Difference in withdrawal thresholds between presurgery and a specific day postsurgery was tested using Wilcoxon signed-rank test. Two-way ANOVA involving the factors treatment (saline, lidocaine) and postoperative day was used to test the significance of differences in withdrawal thresholds between experimental conditions. A probability of 0.05 was chosen as the criterion for significance. The Bonferroni method was used when necessary to correct the probability level for multiple comparisons.
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RESULTS |
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In general, all rats appeared in good health throughout the testing period. They gained weight and exhibited no self-inflicted wounds. The level of general activity was normal during the testing period after surgery.
Mechanical hyperalgesia in L5 DRG-compressed rats
In 13 rats, the L5 DRG was compressed with a 0.7-mm rod and perfused with normal saline at a low flow-rate (1 µl/h) for the first postoperative week. Figure 2 shows results for 3 days before and 1, 7, 14, 21, and 28 days after surgery for the average percent withdrawal responses across animals (n = 13) plotted as a function of stimulus magnitude of the mechanical probe. After compression of the DRG, the response curves shifted leftward of the prelesion curves for both ipsilateral and contralateral hind paws, suggesting a decreased withdrawal threshold. The percent withdrawal responses as a function of the stimulus magnitude were exhibited as an S-shaped curve before surgery; however, after DRG compression, the force-response curves became hyperbolic, indicating an increased cutaneous sensitivity to mechanical stimulation (i.e., leftward shift of the sensitivity threshold).
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The withdrawal thresholds obtained from the S50 using the Hill equation for all 13 rats markedly decreased after DRG compression. Preoperative withdrawal threshold on the ipsilateral hind paw, averaged for the 3 days of testing, was 55.8 ± 2.0 mN; this threshold decreased significantly to 33.8 ± 3.6 mN on the first postoperative day (P < 0.05, Wilcoxon signed-rank test). Postoperative threshold was lower than preoperative threshold for the entire postoperative testing period of 28 days (P < 0.05, Friedman repeated measures ANOVA on ranks; Fig. 3A).
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The contralateral hind paw on the first postoperative day exhibited a significantly decreased sensitivity to mechanical stimuli (hypoalgesia) in 6 of 13 rats tested (before surgery: 57.3 ± 4.2 mN; postoperative day 1: 71.2 ± 6.3 mN; P < 0.05, Wilcoxon signed-rank test, n = 6). This hypoalgesia disappeared in all rats by day 3 after surgery. Despite the initial hypoalgesia, the withdrawal threshold on the contralateral foot decreased significantly from 56.4 ± 2.2 mN before surgery, to 45.5 ± 3.6 mN on the 3rd postoperative day (P < 0.05, Wilcoxon signed-rank test). The decreased withdrawal threshold on the contralateral foot lasted for more than 4 wk after surgery (P < 0.05, Friedman repeated-measures ANOVA on ranks; Fig. 3A).
Mechanical hyperalgesia was at its maximum during the 2nd postoperative week (postoperative day 13) for both hind paws, at which time withdrawal thresholds decreased 40% for the contralateral (56.4 ± 2.2 vs. 0.33.6 ± 2.2 mN), and 65% for the ipsilateral foot (55.8 ± 2.0 vs. 19.4 ± 3.0 mN) from prelesion threshold. After postoperative day 15, thresholds recovered slightly yet never returned to prelesion levels. Reduction in the withdrawal thresholds was greater on the ipsilateral than contralateral hind paw throughout the period of testing (P < 0.05, two way repeated measures ANOVA; Fig. 3A). Postoperative differences in withdrawal threshold between contralateral and ipsilateral hind paws averaged 15.2 ± 2.7 mN with a maximum difference of 23.6 ± 4.8 mN in the first postoperative day and a minimum of 9.8 ± 2.2 mN during the last day of the testing. However, bilateral differences in the withdrawal threshold did not change significantly after day 5 until the end of testing period (P > 0.05, Friedman repeated-measures ANOVA on ranks).
In eight rats, a high flow-rate (8 µl/h) saline pump was used. Behavioral measures revealed that changes in the withdrawal threshold after DRG compression in high flow-rate saline-treated rats were similar to the low flow-rate saline-treated rats in that both the ipsilateral and the contralateral feet developed profound mechanical hyperalgesia (with initial hypoalgesia only on the contralateral hind paw) after DRG compression (Fig. 3B).
In eight normal, unoperated rats, withdrawal thresholds to mechanical stimulation of the hind paws were measured for 28 days following the same procedure as described above. No significant change in the withdrawal threshold was observed in any rat throughout the testing period (P > 0.05, Friedman repeated-measures ANOVA on ranks; Fig. 4).
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Tactile allodynia induced by DRG compression
Preoperatively, none of the 13 rats implanted with the low flow-rate saline pumps responded with quick foot withdrawals to any of the six strokes of a cotton wisp applied to either foot. As early as 1st day postoperatively, two rats exhibited tactile allodynia on the ipsilateral paw, but most rats developed quick responses to light strokes after day 5 that lasted throughout the period of testing (Fig. 5A). Additionally, saline-treated rats with L5 DRG compression developed guarding behavior ipsilateral to the side of the surgery, such as avoidance of weight-bearing and occasionally holding the ipsilateral hind paw in the air in a protected position. The occurrence of licking of the ipsilateral hind paw also suggested the presence of spontaneous pain or allodynia.
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Tactile allodynia was observed on the contralateral paw with a delayed onset and a lower incidence of foot withdrawal (Fig. 5A) that increased over time following surgery. In general, 90% of the rats exhibited a reflex withdrawal to the stroke of a cotton wisp applied to the foot ipsilateral to the compressed DRG. For the contralateral foot, 50% of the rats responded to the cotton wisp application during the period of postoperative testing.
Effects of local perfusion of the DRG with lidocaine on mechanical hyperalgesia
LOW PERFUSION RATE. In this series of experiments, 2% lidocaine was delivered to each compressed ganglion of 18 rats at a flow-rate of 1 µl/h for the first 7 postoperative days. In comparison with prelesion levels, withdrawal thresholds were significantly lower for the ipsilateral hind paw, between days 1 and 17 after DRG compression (P < 0.05, Friedman repeated-measures ANOVA on ranks) in the lidocaine-treated rats. After day 17, withdrawal thresholds to mechanical stimulation recovered toward baseline. For the contralateral hind paw, withdrawal thresholds decreased postoperatively, but to a lesser extent than that observed for the ipsilateral hind paw.
Compared with saline-treated rats, the withdrawal thresholds were significantly higher on the ipsilateral hind paw in lidocaine-treated rats throughout the testing period (P < 0.05, 2-way repeated-measures ANOVA; Fig. 6A). For the contralateral paw, lidocaine treatment did not affect the withdrawal thresholds during the first two postoperative weeks. The mechanical hyperalgesia returned toward to prelesion levels 17 days postoperatively; this was not observed in saline-treated rats, either ipsilaterally or contralaterally. By day 28, the withdrawal threshold on the contralateral foot was significantly higher in lidocaine-treated rats than in the saline-treated ones (P < 0.05, Wilcoxon signed-rank test). Moreover, contralateral hypoalgesia, as occurred in saline-treated rats during first postoperative day, was not observed in lidocaine-treated rats (Fig. 6B).
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HIGH PERFUSION RATE. In high flow-rate (8 µl/h), lidocaine-treated rats (n = 10), withdrawal thresholds were maintained at prelesion levels during the one-day of drug perfusion (first postoperative day). In three of the high flow-rate lidocaine-treated rats, a slight hypoalgesia was observed on the ipsilateral foot during drug infusion (postoperative day 1; Fig. 7). Subsequently, withdrawal thresholds decreased for both ipsilateral and contralateral feet.
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Effects of lidocaine on tactile allodynia
Between postoperative days 1 and 28, the lidocaine treatments significantly reduced the incidence of foot withdrawals to light stroking when compared with saline-treated rats (Fig. 5). As observed for the saline-treated rats, the incidence of foot withdrawal to light stroking in the lidocaine-treated groups of rats was greater in the ipsilateral than contralateral to the DRG-compressed paw (Fig. 5B).
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DISCUSSION |
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This study demonstrated that compression of the L5 DRG induced a central pain syndrome that included bilateral mechanical hyperalgesia and tactile allodynia. Results also suggest that a lidocaine block or reduction of abnormal activity from the compressed ganglia to the spinal cord could prevent the development of mechanical hyperalgesia and allodynia.
The procedure used differs from the DRG compression models reported
previously (Hu and Xing 1998) in three ways. First,
L-shaped rods were used instead of straight ones to prevent the
intrusion of the inserted rod into the spinal cord. Second, the solid
rod was replaced with a hollow, perforated one. And third, the rod was
connected to an implanted pump for the delivery of various chemicals to
the compressed ganglion. This procedure also differs from the one
reported previously, in which both L4 and
L5 lumbar ganglia were compressed with 0.6-mm
solid stainless steel rods (Song et al. 1999
).
In the present study, we used the Hill equation, rather than the logit
transformation as described in our previous studies (Song et al.
1999), to estimate the withdrawal thresholds in normal and
DRG-compressed rat. The major reason for this change was that available
methods used to estimate the withdrawal threshold, such as the up-down
method (Chaplan et al. 1994
), and the method we described previously (Song et al. 1999
), were based on
the assumption that the force-response curve was S-shaped. Thus a
linear logistic transformation could be performed to calculate the 50%
response force. However, we recently have found that compression of the L5 DRG with a 0.7-mm rod significantly increased
mechanical sensitivity, such that in a small number of rats, a
withdrawal response could be evoked by a force as low as 5 mN, the
minimum force employed in our testing procedure. As a result, the
force-response data resulted in a hyperbolic curve, instead of an
S-shaped curve. Therefore logistic transformation would not precisely
estimate the withdrawal threshold for such data. The Hill equation,
however, provides ideal fitting for both S- and hyperbolic shape
response curves and was thus used to precisely estimate the withdrawal thresholds in both normal and neuropathic rats.
MECHANICAL HYPERALGESIA.
Our behavioral results agree with those obtained from the two-ganglion
compressed rat experiments, except that a significant decrease in the
withdrawal threshold to mechanical stimuli was also observed on the
contralateral hind paw. Thresholds in the contralateral hind paw were
higher, and the onset of cutaneous hyperalgesia was slower than in the
ipsilateral paw. Only occasionally in two-ganglion compressed rats was
a change observed in withdrawal threshold on the contralateral foot
(Zhang et al. 1999). In this study, because no surgery
was performed on the contralateral side, the observed changes in
withdrawal threshold on the contralateral foot were not likely related
to the general surgical procedure. Contralateral hyperalgesia is not
novel in animal neuropathic pain models, and it has been reported
previously (Carlton et al. 1994
; Kim and Chung
1992
; Seltzer et al. 1990
).
TACTILE ALLODYNIA.
Another significant finding of this study was that about 50% of rats
with L5 DRG compression developed tactile
allodynia on the contralateral hind paw; this has not been reported in
any previous neuropathic animal model (Bennett and Xie
1988; Kim and Chung 1992
; Seltzer et al.
1990
). Compression of the L4 and
L5 DRG with a solid rod of 0.6 mm in diameter
failed to induce contralateral allodynia. Although the reason for this
is not known, it is likely that development of contralateral
hyperalgesia is related to the diameter of the rod that is used to
compress the ganglion. A smaller diameter (e.g., 0.5-mm) rod tends to
produce less mechanical hyperalgesia with no changes in the sensitivity
on the contralateral hind paw (unpublished observation).
MECHANISMS OF CUTANEOUS HYPERSENSITIVITY AFTER L5 DRG
COMPRESSION.
It is believed that enhanced cutaneous sensitivity to noxious
mechanical stimulation (tactile hyperalgesia) results from central sensitization of the spinal cord, which can develop in human subjects after receiving nociceptive inputs (C-fiber activity) for a period of
time (Torebjork et al. 1992). In neuropathic animal
models, abnormal nociceptive activity is generally thought to be
generated at the injury site of the axons and more proximally from the
DRG somata (Babbedge et al. 1996
; Burchiel
1984
; Czeh et al. 1977
; Hu and Xing
1998
; Kajander et al. 1992
; Wall and
Devor 1983
; Xie et al. 1995
; Zhang et al.
1997b
). Spontaneous activity has been recorded from compressed,
C-type DRG neurons (Zhang et al. 1999
) and is believed
to contribute to the development of mechanical hyperalgesia.
LOCAL PERFUSION OF THE COMPRESSED DRG WITH LIDOCAINE.
Our results indicate that a short period of application of lidocaine
locally to the compressed ganglion, and beginning at the time of
surgery, partially prevented the development of mechanical hyperalgesia
and significantly blocked tactile allodynia. These effects were present
for a long postoperative period, and lasted well beyond the termination
of lidocaine perfusion. Results from the present study are consistent
with previous findings in rats with chronic constriction of the sciatic
nerve in that lidocaine pretreatment of the injured nerve abolished
paw-licking reflex for a variable postoperative period (1 wk or more)
and shortened the duration of thermal hyperalgesia. Our results also
support the finding that a prolonged application of local anesthetics may prevent late development of cutaneous hyperalgesia as reported by
Kissin et al. (1998).
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ACKNOWLEDGMENTS |
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The authors thank Dr. Robert H. LaMotte, Yale University, for help in designing drug-perfusing systems.
This research study was supported by a Pilot Study Fund from the University of Arkansas for Medical Sciences.
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FOOTNOTES |
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Address for reprint requests: J.-M. Zhang, Dept. of Anesthesiology, Slot 515, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: ZhangJunming{at}exchange.uams.edu).
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 14 February 2000; accepted in final form 4 May 2000.
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REFERENCES |
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