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1 From the Department of Ophthalmology, University of Helsinki, Finland; 2 The Netherlands Ophthalmic Research Institute, Amsterdam; 3 Instituto de Neurosciencias, Universidad Miguel Hernandez-CSIC, San Juan de Alicante, Spain; the 4 Department of Ophthalmology, University of Rome "Tor Vergata," Italy; 5 Helsinki City Police Department, Finland.
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Abstract |
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METHODS. Ten police officers who volunteered for the study were exposed to OC. Clinical signs were assessed. Corneal sensitivity was measured using a Cochet–Bonnet or a noncontact esthesiometer that provides separate measurements of mechanical, chemical, and thermal sensitivity. Tear fluid nerve growth factor (NGF) was measured. Corneal cell layers and subbasal nerves were examined by in vivo confocal microscopy. The subjects were examined before application and 30 minutes, 1 day, 1 week, and 1 month after OC exposure.
RESULTS. OC spray produced occasional areas of focal epithelial cell damage that healed within 1 day. Each eye showed conjunctival hyperemia and in two subjects, mild chemosis. All except one eye had unchanged best corrected visual acuity (BCVA). A transient decrease (day 1) of mechanical sensitivity was observed with the Cochet–Bonnet esthesiometer. With the gas esthesiometer, mechanical sensitivity remained below normal values for 7 days. Chemical sensitivity to CO2 was high for as much as 1 day and decreased below normal 1 week later, whereas sensitivity to cold was unaffected. Two subjects had measurable tear NGF that increased after exposure. Basal epithelial cell morphology suggested temporary corneal epithelial swelling, whereas keratocytes, endothelial cells, and subbasal nerves remained unchanged.
CONCLUSIONS. Although OC causes immediate changes in mechanical and chemical sensitivity that may persist for a week, a single exposure to OC appears harmless to corneal tissues. The changes are possibly associated with damage of corneal nerve terminals of mainly unmyelinated polymodal nociceptor fibers.
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Introduction |
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Capsaicin (8-methyl-vanilyl-6-nonenamide), the pungent component of chili peppers, has been shown to induce intense pain in humans and pseudoaffective pain reactions in animals when applied to the skin and the front of the eye, as well as neurogenic inflammation due to the release of neuropeptides contained in nerve terminals.24 25 26 Capsaicin’s effects are associated with acute stimulation of primary sensory nerve endings,27 28 which is accompanied by a depletion of their neuropeptide content.29 30 This process is followed by nerve inactivation and suppression of neurogenic inflammation in response to injury.29 31 32 Capsaicin also has long-lasting effects on sensory nerves and their target tissues. Neonatal injection of capsaicin into small rodents induces a selective sensory denervation followed by a slow and incomplete regeneration of the sensory fibers that is not fully compensated by increased sprouting of intact nerve fibers and persists in adult animals.33 34 35 36 37 Corneal and/or skin ulcers and scars are concomitantly formed and may persist for months,29 34 35 37 38 resembling the clinical picture of neuroparalytic–neurotrophic keratitis secondary to trigeminal denervation.39 Capsaicin treatment in adult animals causes less prominent but still detectable alterations of sensory innervation. In vitro and in vivo studies show that nerve growth factor (NGF) reverses the decrease of transmitter content and restores the peripheral function of primary afferent neurons impaired by capsaicin treatment.40 41 In humans, the effects of capsaicin on sensory innervation are poorly known. Capsaicin injected subcutaneously produces acute pain and hyperalgesia.42 Topically applied capsaicin ointment (0.075%) used for pain relief results in a reduced sensitivity to cutaneous stimuli and decreased numbers of epidermal nerve fibers. Reinnervation of the skin and recovery of sensitivity have been observed after discontinuation of treatment.26
In the present work, we studied in human eyes the effects of topical capsaicin on corneal innervation and corneal sensitivity using in vivo confocal microscopy43 44 and noncontact esthesiometry.45 We also measured tear fluid NGF concentrations in subjects exposed to capsaicin.46
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Methods |
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Subjects and Tests
The Police Department of the Ministry of Internal Affairs and the
Ethical Review Committee of Helsinki University Eye Hospital approved
the research plan, which followed the tenets of the Declaration of
Helsinki. Each subject gave informed consent.
The study was performed in two stages. For the first, five police officers were used (men, 27–34 years of age). All had been exposed several times to OC or to diorthochlorobenzyldenemalononitrile (CS): three officers 3 times, one 5 times, and one 15 times. For the test, subjects were treated with the OC spray and after first aid, were queried about symptoms and examined by slit lamp biomicroscopy, corneal photography, corneal sensitivity testing with a Cochet–Bonnet esthesiometer, determination of tear fluid NGF levels, and in vivo confocal microscopy. The group was re-examined 1 day, 1 week, and 1 month after OC exposure. Visual acuity was also measured at these times. In this group of subjects, prominent changes in corneal sensitivity were noticed. Therefore, in a second group of five police officers (four men and one woman, 24–50 years of age), the eyes were evaluated before and after OC exposure by the same clinical explorations made in the previous group. Corneal sensitivity was, however, more extensively explored using a noncontact gas esthesiometer. Subjects were examined within the first hour, 1 day, and 1 week after OC exposure. Three subjects of this group had never been exposed before to OC or CS. One had undergone bilateral photorefractive keratectomy 2 years earlier. The remaining two officers had experienced two and three previous OC exposures, respectively.
Symptom Query and Scoring of Ocular Lesions
The severity of the symptoms including nasal congestion, dyspnea,
facial hyperemia, and ocular and facial stinging pain were evaluated.
Heart rate was measured with a pulse meter (Polar Electro, Oulunsalo,
Finland) before and 1 and 10 minutes after exposure. Ocular lesions
were scored according to Draize’s scale.47
Testing of Corneal Sensitivity
Contact Sensitivity.
In the first group of subjects, the sensitivity of the central cornea
and of four corneal quadrants was explored in both eyes, by using a
Cochet–Bonnet esthesiometer provided with a number 8
filament.48
Each area was touched three times, beginning
at a filament length of 60 mm and reducing it sequentially in 5-mm
steps. A minimum of two positive answers were required for the response
to be considered positive. The longest filament that evoked the
positive response was considered as the threshold for mechanical
sensitivity.
Noncontact Gas Esthesiometry.
Corneal sensitivity of the second group of police officers was tested
unilaterally with a gas esthesiometer that performed selective
mechanical, chemical, and thermal stimulation of the
cornea.45
Gas jets of 3 seconds’ duration were applied to
the corneal surface at 2-minute intervals. Mechanical stimulation
consisted of a series of pulses of warmed air at flow rates varying
from 0 to 300 ml/min. Chemical stimulation was performed with series of
six pulses of a warmed mixture of air and CO2 at
different concentrations (0–80%). For selective thermal stimulation
10 pulses of air, warmed or cooled in the tip of the probe (from
-10o to +80°C, corresponding to changes in
corneal surface temperature between -5°C and +3°C around the
control value of 34.5°C) were used. At least one blank stimulus (a
pulse with no gas flow) was applied between pulses of each series. To
prevent mechanical stimulation during selective chemical and thermal
stimulation, flows below mechanical threshold value previously measured
for each subject were used. To avoid changes in basal corneal
temperature during selective mechanical and chemical stimulation, the
gas stream was heated up to 50°C at the tip of the
probe.45
Subjects were seated comfortably in front of a slit lamp, with the head supported by the head holder. With the slit lamp table controls, the tip of the gas esthesiometer was adjusted at a distance of 5 mm perpendicular to the center of the cornea. The subject was asked to blink immediately before the stimulus. A click produced by the opening of the valve in the probe identified the onset of the stimulus. Selective mechanical, chemical, and thermal stimulation was performed in the left eye in each session. The protocol was completed after the third session, performed 1 week after OC exposure.
Immediately after each stimulation pulse, the subject had to judge and mark the magnitude of the various parameters of the sensation in a continuous horizontal scale of 10 cm (the visual analog scale [VAS]). In the VAS, 0 was assigned to no sensation and 10 to the maximal sensation ever experienced. Six different components of the sensation were studied: 1) intensity 2) degree of irritation, 3) magnitude of burning pain, 4) magnitude of stinging pain, 5) cooling component of the sensation, and 6) warming component of the sensation (Acosta MC, Belmonte C, Gallar J, unpublished observations).
Tear Fluid Collection
Unilateral tear fluid samples were collected with a scaled 5- or
25-µl fire-polished microcapillary tube, as previously
described.49
The tear fluid flow in the collection
capillary (in microliters per minute) was calculated by dividing the
volume of the tear fluid sample by the collection time. The release (in
picograms per minute) was calculated by multiplying the concentration
in the sample by the tear fluid flow in the collection
capillary.50
As capsaicin exposure induced a marked
hypersecretion of tears, the use of the parameter "release"
(flow-corrected concentration) enabled the comparison of pre- and
postexposure concentrations. The tear samples were directly transferred
to tubes (Eppendorf, Fremont, CA) and stored at -70°C.
Tear NGF Determination
The tear NGF concentrations were measured by a two-site,
immunoenzymatic assay that recognizes human and murine NGF and is
capable of detecting as low as 5 pg/ml.46
Briefly,
polystyrene 96-well microtube immunoplates (NUNC, Napierville, IL) were
coated with affinity-purified polyclonal goat anti-NGF antibody.
Parallel wells were coated with preimmune goat IgG for evaluation of
the nonspecific signal. After overnight incubation at room temperature
and 2-hour incubation with the coating buffer (0.05 M carbonate buffer
[pH 9.5] in 2% bovine serum albumin), plates were washed with 50 mM
Tris-HCl (pH 7.4), 200 mM NaCl, 0.5% gelatin, and 0.1% Triton X-100).
After extensive washing, the diluted tear and NGF standard solutions
were distributed into the wells and left at room temperature overnight.
The plates were washed and incubated with 4 mU/well
anti-ß-NGF-galactosidase (Boehringer Mannheim, Mannheim, Germany) for
2 hours at 37°C and, after another washing, 100 µl of substrate
solution (4 mg chlorophenol red/ml substrate buffer; Boehringer
Mannheim) was added to each well. After an incubation of 2 hours at
37°C, optical density was measured at 575 nm using an enzyme-linked
immunosorbent assay reader (Dynatech Laboratories, Billingshurst, UK),
and the values of standards and samples were corrected by subtracting
the background value produced by nonspecific binding. Data were
expressed in picograms per milliliter, and all assays were performed in
duplicate.
In Vivo Confocal Microscopy
A tandem scanning confocal microscope (TSCM, Model 165A; Tandem
Scanning, Reston, VA) was used to examine all layers of the central
cornea. The left eye of each subject was explored. First a drop of
topical anesthetic (benoxinate hydrochloride; Oftan Obucain;
Santen, Tampere, Finland) was applied on the cornea, and a drop of
2.5% hydroxymethylcellulose gel (Goniosol; IOLAB Pharmaceuticals,
Claremont, CA) was placed on the tip of the objective lens. The setup
and operation of the confocal microscope has been described
previously.43
44
Briefly, a x24, 0.6 numeric aperture
variable working-distance objective lens was used. The field of view
with this lens is 450 x 360 µm, and the z-axis
resolution is 9 µm. Images were detected using a low-light-level
camera (VE1000; Dage, Michigan City, IN) and recorded on SVHS tape.
Video images of interest were digitized using a computer-based imaging
system with custom software (University of Texas Southwestern Medical
Center, Dallas), and printed (Stylus Color 800 printer; Seiko Epson,
Nagano, Japan).
Statistical Analyses
Data are expressed as means ± SEM or SD. Differences between
groups or subjects were examined with parametric (Student’s
t-test, one-way analysis of variance [ANOVA]) or
nonparametric statistical tests (repeated measures, ANOVA on ranks,
Friedman’s test), as necessary. Pearson correlation was used to
determine the stimulus–response relationship. Statistical significance
was set at P < 0.05.
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Results |
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Scoring of Ocular Lesions by Slit Lamp Biomicroscopy
Draize’s scale for scoring of ocular lesions includes signs in
cornea, iris, and conjunctiva.47
Six corneas of four
police officers showed focal corneal epithelial cell damage at 20
minutes as shown in Figure 1
, but none of the corneas showed opacities as described in Draize’s
scale.47
The following day, the epithelial surface of all
subjects was normal again. All eyes showed conjunctival hyperemia
(score 1) at 20 minutes. The mean duration of conjunctival injection
was 9.8 hours (range, 2–24 hours). Mild chemosis (score 1) was
observed in two subjects after the exposure, but it was undetectable
on the following day.
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20/20)
throughout the study. One police officer lost one line at both 1 day
and 1 week after OC.
Sensitivity Testing
Table 1
illustrates that 20 minutes after OC exposure mechanical
sensitivity explored with the Cochet–Bonnet esthesiometer in the first
group of subjects was markedly decreased in all quadrants of the
cornea. One subject had complete bilateral corneal anesthesia, while
reduced mechanical sensitivity was observed in six eyes of the
remaining four police officers. Within 1 day, normal levels were
recovered, remaining normal to Cochet–Bonnet exploration up to 1
month. The sensitivity of right and left eyes at different time points
are shown separately.
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Thirty minutes after exposure to OC, sensitivity to heat had disappeared in two subjects, but threshold for hot stimulation of the remaining three was normal (Table 2 , Fig. 2C ). Similar results were obtained 1 day and 1 week after capsaicin (Table 2 , Fig. 2C ). Cold sensitivity and cold threshold were unaffected by OC (Table 2 , Fig. 2D ).
Stimulus–Response Curves.
Mechanical Stimulation.
Figure 3A
illustrates the stimulus–response curve of subjective intensity of
mechanical stimulation in control conditions and 30 minutes after OC.
In both cases, subjective intensity was significantly correlated with
the magnitude of the stimulus (correlation coefficients: 0.950 and
0.966; P = 0.00,008 and 0.002 for control and 30
minutes after OC exposure, respectively). However, the values of
intensity of the sensation reported for increasing stimulus forces were
lower 30 minutes after OC (Fig. 3A) . Thus, the power of the function
describing the stimulus–response relation (Steven’s power function)
was slightly smaller for OC-treated eyes (exponent: 1.20 versus 1.54 in
control; Fig. 3A
, inset). One day and 1 week after capsaicin, the
intensity curve for mechanical stimulation still deviated to the right
in comparison with control conditions (Figs. 4A
,
5 A).
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Cold Air.
Intensity–response curves obtained with cold stimulation of the cornea
were not modified by OC exposure (Figs. 3D
4D
5D) .
Tear Fluid NGF
NGF was at measurable levels in the tear samples of 2 of 10 police
officers (Figs. 6A 6B
). Of note, these eyes had not had earlier contacts with tear
gases, nor had they undergone any corneal surgery.
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Discussion |
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This study shows that the structural and functional effects of OC spray on conjunctiva and cornea are mild and temporary. This interpretation applies only to the preparation used in the present work; other solvents, such as 92% trichloroethylene, may induce severe corneal damage (Tervo et al., unpublished observations, 1999). Several studies on the ocular effects of capsaicin in animals have been published.27 28 34 38 However, only recently the effects of OC sprays on the human eye were briefly reported.52 These researchers described transient and moderate conjunctival and corneal changes with occasional punctate epithelial erosions. In the present study, mild and short-lasting corneal and conjunctival signs were also observed. It is possible that epithelial damage was caused by the alcohol solvent rather than by capsaicin, because similar grayish white epithelial changes can occasionally be seen after contact with isopropyl alcohol-cleaned, but wet, tonometer tips. Visual acuity was unaffected in all but one eye, in agreement with the data of Zollman et al.52
The acute effects of capsaicin on the sensory activity of corneal nerve afferents in animals are well documented.27 28 53 In the cat eye, capsaicin at high concentrations (1%, 33 mM) produced a transient excitation of polymodal nociceptive fibers followed by inactivation of most fibers to all subsequent stimuli; cold sensory fibers were weakly activated, but many remained active after capsaicin, whereas pure mechanosensory fibers were largely unaffected by this substance. The excitatory effects of capsaicin are due to activation of a vanilloid receptor (VR1) present in polymodal nociceptive neurons54 55 that acts as a nonselective cationic channel, thus depolarizing sensory nerve terminals. This strong excitatory effect on polymodal nociceptive fibers explains the intense pain experienced immediately after capsaicin application to the eye. Toxic effects of capsaicin are the consequence of a massive calcium entrance that leads to cell damage and functional inactivation, making nerve endings insensitive to further stimuli.56
Determinations of mechanical threshold with the Cochet–Bonnet esthesiometer after capsaicin treatment showed an acute reduction of corneal mechanical sensitivity followed by a progressive recuperation of control values.52 Nevertheless, the Cochet–Bonnet esthesiometer does not permit evaluation in detail of the degree of short- and long-term functional disturbances caused by capsaicin in the various populations of sensory fibers that sustain corneal sensitivity. Graded measurement of the responsiveness to mechanical, chemical, and thermal stimulation of the cornea with the gas esthesiometer indicate that the effect of capsaicin on the different populations of corneal nerve fibers was heterogeneous and evolved with time.
With the gas esthesiometer, the reduction of mechanical sensitivity observed with Cochet–Bonnet stimulation was confirmed. Responses to low and moderate mechanical stimulation were depressed to a varying degree among individuals 30 minutes after OC and remained below control values 1 week after OC treatment. Acute blockade of a fraction of polymodal nociceptors, preferentially those with unmyelinated axons that are highly sensitive to capsaicin,53 seemed to be responsible for the immediate reduction of sensitivity to mechanical stimulation. Residual mechanical sensitivity in the first hour after OC application is attributable to activation of pure mechanosensory fibers that have a higher threshold and would be much less affected by capsaicin27 and to those polymodal units presumably A-delta that remained functional. The gradual return of mechanosensitivity during the ensuing hours and days may be ascribed to the recovery of those corneal polymodal fibers that were initially inactivated by capsaicin. The response to chemical stimulation with CO2, which is also mediated by polymodal fibers was enhanced immediately after OC application. Twenty-four hours later, it remained high in three subjects but was absent in two, reappearing in a depressed state in all subjects 7 days after treatment. These results confirm that a variable fraction of the polymodal fibers was acutely inactivated and that this process reached a maximum 24 hours after OC exposure. They further indicate that the fibers that remain functional became sensitized57 thus producing a hyperalgesic response. This phenomenon was more prominent with CO2 than with heat stimulation, although both stimuli activate polymodal nociceptive fibers, probably reflecting the fact that heat responses are mediated by VR1 (capsaicin) receptors while additional, capsaicin-insensitive ion channels participate in the responses to acid.58 The absence of changes in cold sensitivity during the 24 hours after OC exposure indicates that cold-sensory fibers were not immediately affected by capsaicin. Nevertheless, as occurred with the other modalities of sensation, cold sensitivity was depressed 1 week later, implying that a part of both polymodal and cold fibers were disturbed in the long term by the treatment.
NGF is the prototypical member of the neurotrophin family of growth factors.59 It plays a critical role in the development of primary sensory neurons during embryonic life,60 61 including those that innervate the cornea.62 In adult animals, NGF receptors (TrkA) remain in the subpopulation of small nociceptive sensory ganglion neurons.63 During chemically induced inflammation with carrageenan64 or turpentine,65 increased tissue levels of NGF have been measured. Tissue NGF seems to increase the sensibility of peripheral terminals to noxious stimuli.66 In the present experiments two of the five police officers showed detectable levels of NGF in tears, and levels increased after OC treatment. Elevated values could still be measured 1 week later. In spite of the limited number of data, these results suggest that in the cornea,67 as in other tissues, NGF is released during noxious stimulation contributing to sensitization and hyperalgesia of inflamed ocular tissues. Moreover, elevated NGF levels may contribute to nerve sprouting and enhanced neuropeptide synthesis observed in the skin after injury and in the cornea after capsaicin treatment.36 37 This in turn would facilitate healing of the injured target tissues.38 68 69
In vivo confocal microscopy is a noninvasive method for examining tissue responses in different corneal sublayers of the human cornea.43 44 Toxicity of various substances has been evaluated in an animal model,51 but to our knowledge, this is the first in vivo confocal microscopy study on potential toxic effects of a substance on the human cornea. The results show that OC spray causes surface epithelial damage of short duration in some subjects. That the cell borders of the basal epithelial cells were easily visualized at 30 minutes and 1 day after OC, without signs of cell damage, suggests epithelial swelling.
No changes could be ascertained in the morphology of subbasal nerves after a single pepper spray exposure. Electron microscopic observations70 71 have revealed that corneal subbasal nerves that are seen by in vivo confocal microscopy correspond to nerve bundles, because visualization of individual nerve fibers is beyond the level of resolution of confocal microscopy. In most cases the nerves were more apparent after OC exposure, probably because of swelling of the epithelial cells through which the nerves are pressed into the same focal plane. The images of the nerve fiber bundles did not vary during the study, and no signs of sprouting were apparent. It is possible that the insult to the nerves is not great enough to induce sprouting. Alternatively, sprouting may be beyond the level of resolution or is limited to the peripheral cornea, as described for experimental animals,37 which was out of the range of observation in the present experiment in which explorations were limited to the central cornea. A surprising finding was the spirallike nerve fiber bundle arrangement of the subbasal plexus in the eyes of a police officer repeatedly exposed to OC or CS. A similar organization has been observed in the nerves of the cornea of an alkaline phosphatase transgenic mice (Belmonte and Raviola, unpublished observations, 1999), but its significance is obscure.
Because of the mild and transient signs of tissue injury, it can be concluded that single exposure of human eyes to OC is relatively harmless to the cornea and conjunctiva. However, one should be cautious in repeated OC exposures, because long-lasting changes in corneal sensitivity could occur. These changes are possibly associated with damage of nerve terminals of mainly unmyelinated polymodal nociceptive fibers.
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Acknowledgements |
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Footnotes |
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Submitted for publication December 28, 1999; accepted February 15, 2000.
Commercial relationships policy: P (JG, CB); N (MV, LM, AL, JM, TH, TT).
Corresponding author: Minna Vesaluoma, Department of Ophthalmology, University of Helsinki, PO Box 220, FIN-00029 HUS, Finland. minna.vesaluoma{at}hus.fi
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