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Vol. 285, Issue 2, 533-538, May 1998
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas
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Abstract |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The present study investigated whether spinal administration of
S-(+)-3-isobutylgaba (S-(+)-3-IBG) or its
stereoisomer, R-(
)-3-isobutylgaba (R-()-3-IBG), are effective in reducing the
hyperalgesia and swelling observed after injection of kaolin and
carrageenan into the knee joint of the rat. The effects of pretreatment
and post-treatment of S-(+)-3-IBG,
R-()-3-IBG and artificial cerebrospinal fluid (aCSF)
on the swelling, pain-related behavior scores and the heat hyperalgesia
induced by knee joint inflammation were compared. Infusion of either
S-(+)-3-IBG or R-()-3-IBG through a
microdialysis fiber, implanted in the dorsal horn of the spinal cord,
for 1.5 h before injection of kaolin and carrageenan resulted in a
20 to 30% reduction in joint swelling compared with aCSF-treated controls, and prevented the development of heat hyperalgesia and spontaneous pain. In contrast, infusion of either stereoisomer after
the development of inflammation reduced the hyperalgesia but did not
reduce the amount of joint swelling compared with aCSF-treated animals.
In summary, S-(+)-3-IBG and R-()-3-IBG are effective antihyperalgesic agents when administered both before and
after joint inflammation. In addition, if administered before injection
of kaolin and carrageenan into the knee joint this drug can attenuate
joint inflammation. Both the antihyperalgesic and anti-inflammatory
properties of this drug probably are mediated through a central
neurogenic mechanism.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Acute
experimental arthritis can be induced by injection of kaolin and
carrageenan into the knee joint (Sluka and Westlund, 1993a
). The
inflammatory agent, carrageenan, causes plasma extravasation and edema
after the release of neuropeptides (Lam and Ferrell, 1993) and other
inflammatory mediators (Herbert and Schmidt, 1992; Birrell and McQueen,
1993; Birrell et al., 1993) into the joint cavity.
Concomitant with the injury to the joint tissue, both peripheral and
central sensitization occurs (Coggeshall et al., 1983;
Schaible and Schmidt, 1985, 1988; Schaible et al., 1991; Dougherty et al., 1992). This peripheral and central
sensitization is manifested in the awake rat as hyperalgesia (Sluka and
Westlund, 1993a), which can be quantified easily by measuring a
reduction in paw withdrawal latencies to a noxious radiant heat source.
Hyperalgesia induced by injection of kaolin and carrageenan in the rat
can be blocked by the spinal administration of antagonists of the
GABAA receptor, NMDA receptor, non-NMDA
excitatory amino acid receptors and neurokinin 1 receptors (Sluka and
Westlund, 1993b; Sluka et al., 1993, 1994, 1997; Rees
et al., 1995). Both secondary hyperalgesia and allodynia are
mediated by changes within the central nervous system as a result of
increased afferent barrage from the site of injury. Thus, it is thought
that in this model of secondary hyperalgesia GABA, NMDA, non-NMDA and
neurokinin 1 receptors are involved in the maintenance of central
sensitization. Spinal administration of either a
GABAA or non-NMDA excitatory amino acid receptor
antagonist before the injection of kaolin and carrageenan
(pretreatment) results in a reduction in the amount of swelling as well
as in the amount of hyperalgesia observed 4 h after injection
(Sluka and Westlund, 1993b; Sluka et al., 1993). It has been
proposed that this reduction in swelling is caused by the blockade of
DRR, because GABAA and non-NMDA antagonists can
prevent the generation of DRR induced by knee joint inflammation (Rees
et al., 1995). DRR are action potentials fired
antidromically back down the primary afferent fibers to the periphery
which are thought to contribute to the release of neuropeptides in the
joint resulting in potentiation of the inflammation and further
afferent activation. Thus, we previously have proposed that DRR are one of the mechanisms involved in the vicious cycle of pain and
inflammation (Sluka et al., 1995). One hypothesis of this
study was that S-(+)-3-IBG may block DRR. If this is the
case, pretreatment with this drug should reduce the amount of swelling
after injection of kaolin and carrageenan.
S-(+)-3-IBG is a more potent analog of gabapentin, an
anticonvulsant currently in clinical use as an add-on therapy in
patients with partial seizures resistant to conventional therapies (see Goa and Sorkin, 1993; Taylor, 1995 for reviews). Although gabapentin originally was designed as a GABA analog, it interacts with neither GABAA nor GABAB receptors
(Bartoszyk and Reimann, 1985), although more recent studies show that
its effects probably are mediated through the
2
subunit of voltage-dependent calcium
channels (Gee et al., 1996). Because recent
electrophysiological studies have shown that voltage-dependent calcium
channels are involved in the development of hyperalgesia after
inflammation (Neugebauer et al., 1996; Nebe et
al., 1997), the second hypothesis of the present study was that
S-(+)-3-IBG could block hyperalgesia after inflammation of
the knee joint.
Therefore, the present study investigated whether spinal administration of S-(+)-3-IBG or its stereoisomer are effective in reducing the hyperalgesia and swelling observed after injection of kaolin and carrageenan into the knee joint of the rat. The S-(+)-3-IBG or its stereoisomer was administered either after the development of inflammation (post-treatment) or before the inflammation was induced (pretreatment). Both isomers of the drug were used to test whether the actions of this drug were stereospecific.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Placement of microdialysis fibers.
All experiments were
approved by the Animal Care and Use Committee at our institution. Sixty
male Sprague-Dawley rats (235-380 g) were anesthetized with sodium
pentobarbital (Nembutal; 50 mg·kg
1
i.p.). A microdialysis fiber [200 µm outside diameter (o.d.), 45,000 MW Cut-off, Hospal AN69] was coated with epoxy resin, except for a
2-mm section. A small midline incision was made in the back at the
level of the last rib. The muscle was then removed from around the T13
vertebra and a hole drilled in both lateral aspects. The microdialysis
fiber then was passed transversely through the dorsal horn of the
spinal cord between lumbar segments L3 and L6 so that the permeable
2-mm section of the fiber lay in the dorsal horn. The microdialysis
fiber was connected to PE20 tubing (Becton
Dickinson, San Jose, CA) which then was tunneled under the skin to the
nape of the neck. The fiber was stabilized with dental cement. aCSF was
pumped through the tubing at a rate of 5 µl·min1 for 1 h before the
PE20 tubing was sealed and the animals allowed to
recover. Once the rats were awake they were examined for motor deficits; any rat which had motor deficits was automatically excluded from the study. No other problems were encountered in animals implanted
with microdialysis fibers by this method. Previous studies in this
laboratory have shown no differences in behavioral scores measured
before and after fiber implantation. Many of the fiber placements were
checked histologically, and no differences in scores attributable to
fiber placement were detected.
Behavioral testing and assessment of arthritis.
As a measure
of heat hyperalgesia animals were tested for paw withdrawal to radiant
heat according to the protocol of Hargreaves et al. (1988).
On the day after fiber placement animals were housed in small lucite
cubicles on an elevated glass plate. Radiant heat was applied to the
plantar surface of the heel of the hindpaw until the rat lifted the
paw. The time at which this occurred was considered the paw withdrawal
latency. Both paws were tested independently at 5-min intervals, for a
total of five trials. A mean of these five readings was used as the
PWL. In the post-treatment group, the animals were tested
before the induction of arthritis in the knee joint (baseline), 4 h postinduction and 1.5 h after drug infusion, i.e.,
5.5 h after induction of arthritis. In pretreatment rats, PWL was measured before administration of any drugs (baseline) and after the drug had been infused for 1.5 h (postdrug) at which time kaolin and carrageenan were injected into the knee joint. PWL was
measured for a final time 4 h after induction of arthritis. The
experimenter was naive to the expected actions of the drug.
) and secondary is used to illustrate that the hyperalgesia was
measured distant to the site of injury (Lewis, 1942).
To estimate the temperature at which the rats withdrew their paws from
the radiant heat source the temperature of the surface of the glass
plate after heating was measured with a calibrated thermistor.
The circumference of the knee joint also was measured with a flexible
tape measure before injection of kaolin and carrageenan (baseline).
After arthritis was induced two independent observers also scored the
rats to note the extent of guarding of the hindpaw of the arthritic
limb. To quantify these changes, the animals were graded by a
subjective pain-rating scale (0-5) modified from the scale described
by Attal et al. (1990) where: 0 is normal, 1 is curling of
the toes, 2 is eversion of the paw; 3 is partial weight bearing, 4 is
non-weight bearing and guarding and 5 is avoidance of any contact with
the hindlimb.
Induction of arthritis.
Rats were anesthetized briefly with
sodium methohexital (Brevital; 60 mg·kg1
i.p.) after the baseline behavioral test (post-treatment group) or
after infusion of the drug (pretreatment group). The knee joint then
was injected with 3% kaolin and 3% carrageenan suspended in sterile
saline (0.1 ml; pH 7.4) and flexed manually until the rat awoke
(approximately 5-10 min).
Administration of drugs.
All drugs were dissolved in aCSF
(pH 7.4, adjusted by bubbling with 95% CO2/5%
O2) and infused through the microdialysis fiber at a rate of 5 µl·min1. Based on
previous in vitro estimates a maximum of about 1 to 10% of
the drug passes from the dialysis fiber into the spinal cord (Sluka and
Westlund, 1993c; Sluka et al., 1997).
)-3-IBG or aCSF. In the post-treatment
group, drugs were infused at concentrations of 0.1, 0.9 and 10 mg·ml1 (n = 6 for each
treatment group). The most effective dose in the post-treatment group,
10 mg·ml1, was used for the
pretreatment group. Thus the pretreatment group received a
single dose of 10 mg·ml1 of
S-(+)-3-IBG, R-()-3-IBG or aCSF
(n = 6 for each treatment group). The drugs were a gift
from Parke-Davis and were synthesized at Parke-Davis Research
Laboratories, a Division of Warner-Lambert (Ann Arbor, MI).
Statistical analysis. A one-way analysis of variance was used to assess whether there was a dose-dependent effect after drug administration. A post hoc t test was carried out when appropriate. A P value of less than .05 was used to indicate significance for all comparisons unless otherwise specified.
The withdrawal response and circumference data were distributed normally (Kolmogorov-Smirnov test). Therefore, to assess the effects of the drug treatment on PWL and circumference after kaolin/carrageenan inflammation, pairwise comparisons with each control were made by paired t tests. Unpaired t tests were used to make comparisons between treatment groups at the same time point. Baseline values were normalized to 100% expressed as means ± S.E.M. for illustrations. Nonparametric tests were used to analyze the behavioral score which is discontinuous. Pairwise comparisons within the same treatment group were made with Wilcoxon's signed-rank test; comparisons between treatment groups were made with the Mann-Whitney U test.| |
Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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At the outset of the experiment the PWL and knee joint circumference were established for each rat. The mean PWL and knee joint circumference were 9.99 ± 0.22 sec (n = 60) and 5.4 ± 0.02 cm (n = 60), respectively. Calibration of the radiant heat source revealed that 9.99 sec after the radiant heat was started the temperature of the glass plate was approximately 50°C.
Effect of intra-articular injection of kaolin and carrageenan. Four hours after injection of kaolin and carrageenan the PWL to noxious radiant heat decreased to 80% of the baseline value (table 1; n = 42), which indicates the presence of secondary hyperalgesia. This decrease was significant (paired t test, P < .01). Calibration of the radiant heat source revealed that after induction of arthritis the rats withdrew the paw of the arthritic limb when the glass plate reached a temperature of 45°C.
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Effect of S-(+)-3-IBG or R-()-3-IBG
infused into the spinal cord after the development of acute
arthritis.
Microdialysis infusion of S-(+)-3-IBG or
R-()-3-IBG 4 h after injection of kaolin and
carrageenan reduced the hyperalgesia to radiant heat normally observed
after inflammation of the knee joint, in a dose-dependent manner
(analysis of variance, P < .03 for each drug; table
2). The PWL values after infusion of the lowest dose of either drug were not different from those recorded 4 h after induction of arthritis (table 2; B A,
i.e., PWL after drug minus PWL at 4 h).
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1, of either isomer of IBG through
the microdialysis fiber 4 h after the knee joint was inflamed
resulted in a return of the PWL to the baseline value (table 2, B A; fig. 1, upper panel), whereas the
PWL obtained with infusion of aCSF remained significantly reduced from
baseline. The effect observed after microdialysis infusion of the 10 mg·ml1 dose of S-(+)-3-IBG
was significantly different from that observed after infusion of the
0.1 mg·ml1 dose (post hoc
t test, P < .02). Furthermore, although there was a
tendency for S-(+)-enantiomer to be more effective at
reducing the hyperalgesia to radiant heat than the
R-()-enantiomer, this difference was not significant
(unpaired t test, P > .1).
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1 concentration the paw
posture was almost baseline, 0 (fig. 1, middle panel).
However, infusion of either S-(+)-3-IBG or
R-()-3-IBG for 1.5 h, 4 h after the development
of acute inflammation, did not reduce the amount of swelling; the
circumference of the knee joint after drug infusion was not
significantly different from the knee joint circumference in animals in
which aCSF was infused (fig. 1, lower panel).
Effect of S-(+)-3-IBG or R-()-3-IBG
infused through the spinal cord before the development of acute
arthritis.
Microdialysis infusion of 10 mg·ml1 of S-(+)-3-IBG or 10 mg·ml1 of R-()-3-IBG or
aCSF through the dorsal horn of the spinal cord alone did not change
the PWL in the heat hyperalgesia test when compared with baseline
values (fig. 2, upper panel).
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1 of S-(+)-3-IBG or
R-()-3-IBG was infused through the spinal cord for
1.5 h before the injection of kaolin and carrageenan (fig. 2, top
panel). Furthermore, pretreatment with S-(+)-3-IBG or
R-()-3-IBG prevented the development of abnormal paw
posture indicative of spontaneous pain (fig. 2, middle panel).
Infusion of S-(+)-3-IBG or R-()-3-IBG through
the spinal cord for 1.5 h before the induction of arthritis
significantly reduced (P < .05; unpaired t test) the
amount of swelling typical after injection of kaolin and carrageenan by
approximately 30%, when compared with rats in which aCSF was infused
through the microdialysis fiber (fig. 2, bottom panel). Although the
S-(+)-enantiomer tended to be more effective at reducing the
amount of swelling than the R-()-enantiomer, the
difference between the two was not significant (unpaired t
test, P > .07).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The results from this study show that injection of kaolin and
carrageenan into the knee joint of the rat results in an acute arthritis which is characterized by secondary heat hyperalgesia, swelling of the knee joint and spontaneous pain. Microdialysis infusion
of S-(+)-3-IBG or R-()-3-IBG through the dorsal
horn of the spinal cord after inflammation of the knee joint reduced the amount of heat hyperalgesia and the spontaneous pain observed in a
dose-dependent manner, but did not alter the amount of swelling of the
knee joint when compared with rats treated with aCSF. Microdialysis infusion of either enantiomer for 1.5 h before the injection of kaolin and carrageenan (pretreatment) did not change the baseline responses to the noxious radiant heat stimulus. However, pretreatment with either enantiomer reduced the amount of swelling observed and
blocked the development of secondary hyperalgesia and spontaneous pain
normally observed after inflammation.
The antihyperalgesic effects of S-(+)-3-IBG are consistent
with other studies. Singh and colleagues (1996) demonstrated that systemic administration of the analog of S-(+)-3-IBG,
gabapentin, reduced thermal hyperalgesia after carrageenan inflammation
of the paw. Other studies have shown that gabapentin also can block hyperalgesia and allodynia in rat models of neuropathic pain (Xiao and
Bennett, 1995). More recently Field et al. (1997)
demonstrated that S-(+)-3-IBG is antihyperalgesic in the
formalin test and in a carrageenan-induced inflammatory pain model.
The antihyperalgesic properties of gabapentin and its analog,
S-(+)-3-IBG, are thought to be centrally mediated. Stanfa
and colleagues (1997) recently demonstrated that carrageenan-induced sensitization of dorsal horn neurons can be blocked by gabapentin. A
more recent behavioral study showed that a dose which blocked hyperalgesia when administered intrathecally was ineffective when administered peripherally (Field et al., 1997). Our study
confirms that the antihyperalgesic property of S-(+)-3-IBG
is, at least in part, centrally mediated because this method of drug
administration, microdialysis infusion into the dorsal horn, has been
shown to limit drug delivery to the spinal cord (Sluka et
al., 1994).
An in vitro binding study showed that S-(+)-3-IBG
and its stereoisomer bind to the 2 subunit
of voltage-dependent calcium channels, the R-()-3-IBG
isomer binding with a 10-fold lower potency than its stereoisomer (Gee
et al., 1996). However, in the present study we did not
observe any differences in potency between the two isomers, although
the S-(+)-enantiomer tended to be more effective. This
result is a little surprising but may be because of the small
difference in potencies between the two drugs and the relatively small
sample size (n = 6). The 2
subunit appears to be common to all voltage-dependent calcium channels (Isom et al., 1994; Hofmann et al., 1994) where
it is thought to increase the expression of calcium channel complexes
(Williams et al., 1992; Brust et al., 1993; Isom
et al., 1994; Gurnett et al., 1996). Thus, the
actions of S-(+)-3-IBG and its stereoisomer may involve more
than one type of voltage-dependent calcium channel. For example,
electrophysiological studies with calcium channel blockers have shown
that both N- and L-type voltage-dependent calcium channels are involved
the development of hyperalgesia after carrageenan-induced inflammation
(Neugebauer et al., 1996). However, blockade of N- and
L-type channels appears to be involved in the generation of pain evoked
by noxious mechanical stimulation in normal tissue as well as in the
mechanical hyperalgesia associated with inflammation (Neugebauer
et al., 1996). More recent work from the same laboratory
shows that blockade of P-type calcium channels has inconsistent effects
upon pain evoked by noxious mechanical stimulation in normal tissue,
but significantly reduces the mechanical hyperalgesia associated with
inflammation (Nebe et al., 1997). Because voltage-dependent
calcium channels are involved in the release of neurotransmitters,
S-(+)-3-IBG and its stereoisomer may prevent the release of
transmitters involved in the process of central sensitization, for
example, glutamate and substance P.
Although a reduction in the amount of swelling was not observed if the
drug was administered after the development of inflammation, administration of the drug before injection of the kaolin and carrageenan significantly reduced the amount of swelling observed at
4 h when compared with aCSF-treated animals. This finding is in
contrast to previous studies with this drug which have not shown a
reduction in swelling after carrageenan-induced inflammation of the paw
(Singh et al., 1996). The route of administration (spinal versus systemic) and the duration of administration
(continuous infusion for 1.5 h versus single dose) as
well as the different model of inflammation may account for this
difference in findings. Again, the 2
subunit of voltage-dependent calcium channels probably is involved in
mediating the anti-inflammatory action of this drug. Blocking the
2 subunit of voltage-dependent calcium
channels may prevent the inward depolarizing current of central primary afferent terminals from reaching threshold. This would prevent the
initiation of DRR which have been shown to be present after inflammation, and are thought to be evoked as a result of excessive depolarization of the central terminals of primary afferent fibers (Sluka et al., 1995). Blockade of these DRRs has been
proposed as a mechanism by which spinally mediated events can control
the amount of swelling induced by injection of kaolin and carrageenan into the knee joint.
Post-treatment with S-(+)-3-IBG or its stereoisomer
antagonized the development as well as the maintenance of heat
hyperalgesia, whereas a reduction in swelling was observed only if the
drug was administered before the onset of inflammation. This implies that the 2 subunit of voltage-dependent
calcium channels are involved in the maintenance of hyperalgesia. In
contrast, the 2 subunits of
voltage-dependent calcium channels probably are involved only in the
initiation and not the maintenance of peripheral inflammation.
In summary, S-(+)-3-IBG and R-()-3-IBG are
effective antihyperalgesic agents, attenuating both secondary
hyperalgesia and spontaneous pain behavior, when administered both
before and after joint inflammation. In addition, when administered
before injection of kaolin and carrageenan into the knee joint, this
drug can attenuate joint inflammation through a central neurogenic
mechanism.
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Footnotes |
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Accepted for publication January 20, 1998.
Received for publication June 4, 1997.
1 This work was supported by National Institutes of Health grant NS 32778.
Send reprint requests to: Karin N. Westlund High, Department of Anatomy and Neurosciences, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1069.
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Abbreviations |
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PWL, paw withdrawal latency;
S-(+)-3-IBG, S-(+)-3-isobutylgaba;
R-()-3-IBG, R-()-3-isobutylgaba;
S.E.M., standard error of the mean;
aCSF, artificial cerebrospinal
fluid;
DRR, dorsal root reflex;
NMDA, N-methyl-D-aspartate;
GABA, 
-aminobutyric acid.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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2 subunit of a calcium channel.
J Biol Chem
271:
5768-57762 subunit in current stimulation and subunit interaction.
Neuron
16:
431-440[Medline].
-Agotoxin IVA, a P-type calcium channel antagonist, reduces nociceptive processing in spinal cord neurons with input from the inflamed but not from the normal knee joint
an electrophysiological study in the rat in vivo.
Eur J Neurosci
9:
2193-2201[Medline].1, 2, 
subunits of a novel neuronal calcium channel subtype.
Neuron
8:
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