Download Reduction of capsaicin-induced ocular pain and neurogenic

Reduction of Capsaicin-Induced Ocular Pain and
Neurogenic Inflammation by Calcium Antagonists
Gertrudis G. Gonzalez, Pilar Garcia de la Rubia, Juana Gallar, and Carlos Belmonte
Purpose. To examine whether blockade of chemosensitivity of corneal nociceptors by Ca2+
antagonists decreases pain and irritation induced by capsaicin.
Methods. In adult rabbits, the number of lid-squeezing movements and the degree of palpebral
opening, miotic response, and conjunctival vasodilation evoked by a bilateral instillation of 30
n\ of capsaicin (33 mM) were measured at different times (up to 5 hours) after the drug.
Irritative responses to capsaicin in eyes pretreated with diltiazem, verapamil, or nifedipine
were compared with those that received only the vehicle. Protein content in aqueous humor
was also measured at the end of the experiment.
Results. Diltiazem at doses of 1 to 28 mM, administered 15 minutes before the application of
capsaicin, significantly decreased scratching movements, conjunctival hyperemia, closure of
the eye, and elevated aqueous protein concentration induced by capsaicin; however, it did not
significantly reduce miosis. Nifedipine (2.8 and 10 mM) diminished the number of scratching
movements but not other inflammatory parameters, whereas verapamil (2.8 and 10 mM) was
totally ineffective in attenuating ocular signs of irritation produced by capsaicin.
Conclusions. These results suggest that by lowering capsaicin-induced neural activity in nociceptive terminals, diltiazem decreases pain and neurogenic inflammation and may be useful as
both an analgesic and an antiinflammatory agent in the eye. Invest Ophthalmol Vis Sci.
1993;34:3329-3335.
W h e n applied topically to the eye of experimental
animals, capsaicin, the pungent substance of the red
pepper, evokes immediate pain as evidenced by lidsqueezing and vocalization, as well as by signs of acute
ocular irritation (conjunctival edema and hyperemia,
miosis, aqueous flare).12 In the human eye, topical
capsaicin at low doses also elicits a sharp sensation of
pain.3 The algesic and inflammatory effects of capsaicin, which are followed by an insensitivity of the area
to chemical irritation, are caused by a selective action
of the drug on polymodal nociceptors, that is, sensory
fibers responding to injurious mechanical forces, irritant chemicals, and heat.4-5
The cornea, conjunctiva, and anterior uvea are
densely innervated by polymodal nociceptive fibers.6
From the Departament of Physiology and the Institute of Neurociences, University of
Alicante, Alicante, Spain.
Supported by Direccion General fnvestigacion Cientifica y Tecnica PM90-0113,
Madrid, and the Centro de Investigaciones Oculares, Alicante, Spain.
Submitted for publication December 1, 1992; accepted April 7, 1993.
Proprietary interest category: PCc5.
Reprint requests: Dr. C. Belmonte, Inslituto de Neurociencias, Universidad de
Alicante, Aptdo. 374, 03080 Alicante, Spain.
Capsaicin acts on polymodal nerve terminals through
the opening of nonselective cationic channels,7 presumably the same ionic channels that mediate response of nociceptors to acid and other irritant chemicals;8'9 this leads to depolarization and the subsequent
discharge of nerve impulses.1011 In addition, Ca2+ influx releases neuropeptides contained in nociceptive
afferents that contribute to local inflammatory reactions (neurogenic inflammation);1213 toxic accumulation of intracellular Ca2+ ultimately determines damage and inactivation of nociceptive terminals.14
Recently, it has been demonstrated that the impulse response of corneal polymodal nociceptors to
certain chemicals (H+, inflammatory mediators) can
be selectively eliminated by blockade with calcium antagonists without affecting mechanical responsiveness
of the same nerve fibers.1516 A suppression of the response of corneal nociceptive terminals to chemicals
may prevent their excitation by capsaicin and the subsequent release of neuropeptides, thus reducing neurogenic inflammation. To explore this possibility, we
studied in rabbits whether calcium antagonists atten-
Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12
Copyright © Association for Research in Vision and Ophthalmology
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3329
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Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12
uated pain and anterior segment irritation induced by
capsaicin administration. A preliminary report of
these findings has been published.17
eyes of the same rabbit were made using parametric
(paired Student's tf-tests) and nonparametric tests
(Wilcoxon's signed rank test).
METHODS
RESULTS
Unrestrained albino California adult rabbits weighing
2.5 to 3 kg were used in this study. The animals were
treated according to the ARVO Resolution on the Use
of Animals in Research.
All animals were administered 30 /xl of a 1% capsaicin solution (33 mM) applied topically to each eye at
a 3-minute interval. In 76 rabbits, 30 ^1 of a calcium
antagonist solution were instilled in one eye 15 minutes before capsaicin, whereas the contralateral eye
was pretreated with the vehicle. A separate, control
group of nine rabbits received the vehicle in both eyes
15 minutes before capsaicin administration.
The number of scratching movements directed to
the eye that received the capsaicin solution was
counted during 1 minute after instillation of the drug.
Palpebral opening in the center of the eye and pupil
diameter were also measured with a transparent ruler
under constant illumination. Palpebral closure was expressed as percent of the distance between lids measured at the beginning of the experiment. Miotic responses were evaluated afterward and expressed as reduction of initial pupillary diameter (in mm) by gently
pulling the upper eyelid to visualize the pupil and the
conjunctiva, when necessary. The severity of conjunctival hyperemia was assessed by biomicroscopy and
quantified by a score from 0 (absence) to 4 (maximal
vasodilation) in eight steps. Measurements were performed at established times during a 5-hour period by
an observer who ignored whether or not eyes had been
pretreated with a Ca2+ antagonist or with the vehicle.
At the end of the experiment, animals received a
lethal dose of intravenous sodium pentobarbital, and
the anterior chamber was punctured to collect
aqueous humor. Protein concentration was determined by Lowry's method.
Capsaicin (Sigma, St. Louis, MO) was dissolved in
15% ethanol and 8.5% Tween-80 and then diluted
with saline to the final concentration (33 mM). Solutions of the calcium antagonists diltiazem (0.1 to 28
mM), verapamil (2.8 and 10 mM), and nifedipine (2.8
and 10 mM) (Sigma) were prepared in isotonic saline
(124 mM NaCl, 5 mM KC1, at pH = 7.5 adjusted with
20 mM HEPES).
When appropriate, data were expressed as the
mean ±SEM of the difference between values in the
eye treated with the Ca2+ antagonist and in the contralateral, vehicle-treated eye. Comparisons between
groups were made with the unpaired Student's Z-test.
Significance was set at P < 0.05. Comparisons between
Irritative Response to Capsaicin
Capsaicin (33 mM) applied bilaterally to the eyes of
nine rabbits elicited a variable number (3 to 16; mean,
100 T
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50 +
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180
240
300
2T
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1 ••
tn
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o
0
•
60
120
180
240
300
60
120
180
240
300
TIME AFTER CAPSAICIN (mln)
FIGURE 1. Temporal course of irritative signs after capsaicin
(33 mM) in nine rabbits pretreated with saline (control
group). Values represent mean ±SEM of nine rabbits. Comparison with pre-capsakin values, paired Student's Mest; *P
< 0.05, +P < 0.005, ++P < 0.001.
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Neurogenic Inflammation and Calcium Antagonists
3331
2,8 mM diltiazem
1 mM diltiazem
0
w
Scratching
Movements
Palpabral
Closure
MIOSIB
Conjunctlval
Hyperemia
-2
Scratching
Movements
Palpebral
Closure
10 mM diltiazem
Mlosls
Conjunctlval
Hyperamla
28 mM diltiazem
C/3
Scratching
Movements
Palpebral
Closure
Mlosls
Conjunctlval
Hyperemia
-2
-8
J
Scratching
Movements
l
++
Palpebral
Closure
Mlosls
Conjunctlval
Hyperemia
FIGURE 2. Differences in irritation signs between diltiazem-treated and contralateral, vehicle-
treated eyes. Number of scratching movements during 1 minute after capsaicin were
counted. Eye closure (mm), miosis (mm), and hyperemia (arbitrary units) were evaluated at 5
minutes, 15 minutes, 75 minutes, 180 minutes, and 270 minutes after capsaicin (consecutive
bars). Data are mean ±SEM of nine rabbits. *P < 0.05, **P < 0.01, +P < 0.005 and ++P <
0.001, paired Student's Mest.
7.1 ± 0.9) of lid-squeezing movements after its application, accompanied by closure of the eyelid followed
by a gradual opening. The pupillary diameter at this
time was markedly reduced; conjunctival hyperemia
also developed within 5 minutes of capsaicin administration (Fig. 1). These signs of anterior segment inflammation subsided gradually. Pupil diameter returned to
normal 2 to 3 hours after capsaicin, but a degree of
palpebral closure and conjunctival hyperemia persisted at least for 4 to 5 hours (Fig. 1). In aqueous
humor samples taken 5 hours after capsaicin administration, aqueous humor protein concentration was significantly elevated (capsaicin: 1.8 ± 0.2 mg/ml, n = 7;
control: 0.7 ±0.1 mg/ml, n = 10, unpaired Student's
Mest, P < 0.005).
signs of ocular discomfort. An absence of reaction was
also observed in the contralateral eye, where the vehicle was applied. When concentrations of diltiazem
above 10 mM were used, a slight conjunctival vasodilation lasting for 5 to 10 minutes was noticed in test eyes.
Mechanical threshold for blinking reflex was not modified after 10 mM diltiazem (control: 4.9 ± 0.3 mg;
after diltiazem: 5.2 ± 0.4 mg; n = 6, not significant).
Effects on the Irritative Response to Capsaicin. In cap-
saicin-treated animals, topical diltiazem administered
15 minutes before capsaicin reduced the signs of pain
and the inflammatory response evoked by the toxin.
This effect is illustrated in Figure 2, where the time
course of pain and irritation parameters are represented for four separate groups of rabbits treated with
increasing concentrations of diltiazem (1 to 28 mM).
Effects of Calcium Antagonist Diltiazem
Although the number of scratching movements, the
Direct Effects. In 41 rabbits, unilateral administration conjunctival hyperemia, and the closure of the eye
were markedly diminished by diltiazem at all doses,
of diltiazem at doses of 0.1 mM (n = 9), 1 mM (n = 9),
prevention of the miotic response to capsaicin was less
2.8 mM (n = 9), 10 mM (n = 9), or 28 mM (n = 5) did
pronounced. Figure 3 depicts the dose-response
not evoke scratching movements or other immediate
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Investigative Ophthalmology 8c Visual Science, November 1993, Vol. 34, No. 12
3332
50 T
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0.0
0.1
2.8
10
1
0.1
28
2.8
10
28
DILTIAZEM (mM)
DILTIAZEM (mM)
FIGURE 3. Dose-dependent inhibitory effect of diltiazem on irritatory reaction to capsaicin 15
minutes after administration of the drug. Each point is the mean ±SEM of 9 to 18 eyes.
Comparisons between eyes of control rabbits (vehicle) and diltiazem-treated rabbits were
made with the unpaired Student's Mest. *P< 0.05, **P < 0.01, +P < 0.005, and ++P < 0.001.
curves for each of the signs of ocular irritation, with
diltiazem concentrations ranging from 0.1 to 28 mM.
Increases in aqueous humor protein concentration
were significantly reduced 30 minutes after capsaicin
instillation. At 5 hours, the difference persisted but
was not significant (Table 1).
Effects of Calcium Antagonists Verapamil and Nifedi-
pine. In 15 rabbits, unilateral treatment with 2.8 mM
(n = 6) or 10 mM (n = 9) verapamil failed to reduce
significantly the signs of ocular pain and irritation elicited by capsaicin when compared with contralateral,
vehicle-treated eyes; moreover, with 10 mM verapamil, conjunctival vasodilation was more pronounced (Table 1 and Fig. 4, upper part).
With nifedipine applied to one eye in 15 rabbits at
2.8 mM (n = 6) or 10 mM (n = 9) concentrations,
palpebral closure, miotic reaction, conjunctival hyperemia, or elevation of protein content in aqueous humor were not prevented (Table 1 and Fig. 4, lower
part). Nevertheless, the higher concentration, 10 mM
nifedipine, reduced the number of scratching movements evoked by capsaicin (Fig. 4D).
DISCUSSION
As in previous reports,1-2 capsaicin, applied topically
to the rabbit eye, evoked immediate signs of pain and
inflammation in the anterior uvea, cornea, and conjunctiva, that subsided gradually within 5 to 6 hours.
These signs of ocular irritation induced by capsaicin
were reduced in a dose-dependent manner by the Ca2+
antagonist diltiazem, whereas nifedipine and verapamil were not effective. The attenuation by diltiazem of irritative effects of capsaicin was more pronounced on corneal pain, conjunctival hyperemia, and
blood-aqueous barrier breakdown than on miosis.
This could be explained in part by a reduced access of
the drug to intraocular structures but also by the different mechanisms postulated for pupillary and vascular responses to ocular injury that involve substance P
and calcitonin gene-related peptide, respectively.18 In
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Neurogenic Inflammation and Calcium Antagonists
3333
l. Protein Concentration in Aqueous
Humor (mg/ml) 30 Minutes and 5 Hours
After Capsaicin in Rabbits Pretreated With
a Ca2+ Antagonist in One Eye and With
the Vehicle in the Contralateral Eye
TABLE
spite of the well-known relaxing effects of diltiazem on
blood vessels of other body tissues,19 conjunctival vasodilation resulting from topical instillation of the
drug was evident only with concentrations more than
10 mM.
Diltiazem is a calcium channel blocker of the
group of benzothiazepines, while nifedipine and verapamil belong respectively to the families of dihydropyridines and phenylalkylamines.20 All of them act at
micromolar concentrations on L-type Ca2+ channels.21
However, diltiazem at higher concentrations (0.5 to 1
mM), also has a blocking action on ionic channels involved in transduction by sensory cells. For instance,
diltiazem reduced the proton-induced sodium current
mediating responses of cultured dorsal root sensory
neurons to acid;22 it also antagonized ionic currents
associated to light transduction in photoreceptors and
responses to odorants of olfactory cells.23 Moreover,
activation by acid of polymodal nociceptive terminals
of the cornea was markedly reduced by diltiazem, suggesting that chemosensitive ionic channels in nociceptors were affected by the drug.15
Capsaicin increases membrane conductance to cations and raises intracellular Ca2+ levels, leading to depolarization and release of neuropeptides by nociceptive terminals.7'9-14 Whether this action of capsaicin on
nociceptors is mediated by the transducing mechanism used by H4" or other chemical irritants is open to
discussion;8-924 however, it is conceivable that the
same ionic channels are finally gated by the various
chemical stimuli for depolarization of polymodal
2.8 mM verapamil
10 mM verapamil
Treatment
Dose
(mM)
Diltiazeni
0.1
1
2.8
10
28
Verapainil
Nifedipine
10*
2.8
10
2.8
10
Treated Eye
Contratateral Eye
AT
1.9 ± 0 . 2
3.4 ± 1.4
2.1 ±0.2
3.7 ±0.9
2.2 ±0.4
2.7 ±0.6
1.5 ±0.2
6.2 ± 0.8
1.3 ±0.2
1.5 ±0.2
2.0 ± 0.4
1.2 ±0.2
9
3
8
1.9 ±0.5
2.5 ± 0.5
1.6 + 0.5
3.7 ± 0.7f
1.0 ±0.2
2.4 ± 0.6
2.1 ± 0 . 9
1.3 ±0.2
8
5
5
6
9
6
9
Values are mean ± SKM.
* 30 minutes after capsaicin.
t / J < 0.01. paired t-tesi.
E
E
1
O 0
3
-i—«i
(D
E-4
Scratching Palpebral
Movements Closure
E
Eo
Miosis
Scratching Palpebral
Movements Closure
Conjunctival
Hyperemia
Miosis
Conjunctival
Hyperemia
10 mM nifedipine
2.8 mM nifedipine
0 d-
-1
E
13
r
-2
-8
Scratching Palpebral
Movements Closure
Miosis
Conjunctival
Hyperemia
t/J
Scratching Palpebral
Movements Closure
FIGURE 4. Effects of verapamil (upper part) and nifedipine (lower part) on the irritative reaction to capsaicin, measured at different times (see legend to Fig. 2). Data are mean ±SEM of
differences between eyes treated with the Ca2+ antagonist and with the vehicle (n = 9). *30
minutes after capsaicin (P < 0.05). + 3.7 ± 0.7 (P < 0.005 paired Student's west).
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Miosis
Conjunctival
Hyperemia
-2
3334
Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12
nerve endings. Our observation that diltiazem, which
blocks nociceptive excitation by acid, also reduced behavioral signs of pain evoked by capsaicin, suggests
that this is the case.
The decreased responsiveness to chemicals of nociceptive nerve endings after diltiazem may also explain the attenuating effect of this drug on capsaicininduced neurogenic inflammation.5 It is well established that inflammation evoked by various types of
ocular injury (chemical irritation, argon laser, or infrared radiation of the anterior segment) is largely dependent on intact sensory nerves that release neuropeptides 18 ' 25 " 27 and can be eliminated by blocking
nerve excitation with local anesthetics or tetrodotoxin.5-28"30 Diltiazem, by diminishing the discharge of
nerve impulses evoked by irritant chemicals and the
entrance of Ca 2+ , presumably decreases the concomitant release of inflammatory neuropeptides. However,
unlike local anesthetic drugs that block completely
nerve excitation or impulse conduction, 31 diltiazem
maintains mechanosensitivity of the same nerve terminals made unresponsive to chemicals. Therefore, diltiazem may be a potentially useful drug to reduce ocular pain and neurogenic inflammation while it preserves corneal sensitivity to mechanical stimuli.
Key Words
cornea, conjunctiva, iris, nociceptors, diltiazem
Acknowledgments
The authors thank S. Moya and A. Perez-Vegara for technical assistance.
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