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Silver nitrate cauterization: characterization of a new model of corneal inflammation and hyperalgesia in rat
Pain 105 (2003) 393–401 www.elsevier.com/locate/pain
Research paper
Silver nitrate cauterization: characterization of a new model of corneal inflammation and hyperalgesia in ratq H.N. Wenk, C.N. Honda* Department of Neuroscience and Graduate Program in Neuroscience, University of Minnesota, 6-145 Jackson Hall, 312 Church Street, Minneapolis, MN 55455-0329, USA Received 11 June 2002; received in revised form 27 August 2002; accepted 6 September 2002
Abstract Chemical cauterization of the central cornea with silver nitrate was assessed as a superficial injury model of tissue sensitization accompanying acute inflammation. Adult male Sprague– Dawley rats were anesthetized with halothane gas, and the centers of their right corneas treated with a silver nitrate applicator stick (75% silver nitrate, 25% potassium nitrate) to produce a discrete lesion 1 mm in diameter. Edema of the corneal stroma and elevated immune cell counts became significant 4 h after cauterization, and were still evident after 48 h. Behavioral sensitization to chemical stimuli was determined by counting the number of blinks following application of 1 mM capsaicin directly to the corneal surface. A significant increase in stimulus-induced blinking was evident 2 h after cauterization. Chemical sensitization peaked at 6 h, and was no longer significant at 12 h. We conclude that silver nitrate cauterization produces acute corneal inflammation and hyperalgesia, and may prove a useful model for the study of primary afferent nociceptors. q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Cornea; Inflammation; Hyperalgesia
1. Introduction The cornea is well suited for the study of primary afferent nociceptors. It is the most densely innervated epithelium in the body, with an innervation density up to 600 times that of skin, and up to 40 times that of tooth pulp (Rozsa and Beuerman, 1982). The cornea contains the same basic types of nociceptors as does skin, but in a simplified, avascular environment. In addition, free nerve endings terminate only a few microns below the corneal surface, allowing for more direct access to sensory nerve terminals and the ability to perform rapid and consistent delivery of topically administered compounds. Chemical cauterization of the murine central cornea by topical application of silver nitrate has been used to study ocular angiogenesis and injury-induced clouding of the cornea (Sunderkotter et al., 1991; Sonoda et al., 1998). However, this model has not been used in behavioral testing q
Supported by NIH grants DA09641 and DE07288. * Corresponding author. Tel.: þ 1-612-624-3915; fax: þ1-612-624-8118. E-mail address: [email protected] (C.N. Honda).
of ocular sensitivity or pain, and has not been used before in rat. The aim of this study is to develop and characterize a silver nitrate model of non-specific inflammation in the adult rat cornea, including behavioral assessment of accompanying changes in pain sensitivity.
2. Methods All experimental procedures were approved by the Institutional Animal Use and Care Committee at the University of Minnesota and conform to established guidelines. 2.1. Induction of inflammation Adult male Sprague – Dawley rats 250 – 300 g were deeply anaesthetized with halothane (4%, in air), and the centers of their right corneas cauterized with a silver nitrate applicator stick (75% silver nitrate, 25% potassium nitrate; Graham-Field Inc, Hauppauge, NY). The applicator was held in contact with the cornea for 2 s, producing a discrete
0304-3959/$30.00 q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0304-3959(03)00295-1
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grayish-white lesion 1 mm in diameter. The cauterized eye was then rinsed several times with room temperature saline, and a thin layer of ophthalmic ointment (Artificial Tears Ointment; lanolin, mineral oil, white petrolatum; Phoenix Pharmaceutical, St. Joseph, MO) was applied to both eyes. Rats recovered fully from anesthesia within a few minutes, and exhibited no outward signs of distress. A few rats kept the cauterized eye slightly closed for up to 4 h following the procedure. 2.2. Histology Both cauterized and contralateral non-cauterized corneas were collected from eight rats at each the following 11 time points after silver nitrate cauterization: 1, 2, 4, 6, 8, 12, 24, and 48 h, and 1, 2 and 4 weeks. In addition, eight naı¨ve rats were used as a control group. Rats were deeply anaesthetized with sodium pentobarbital (65 mg/kg, IP) and perfused through the heart with 0.9% saline at room temperature followed by 4% paraformaldehyde at 48C. Both eyes were removed and immersion-fixed in 4% paraformaldehyde with 0.4% picric acid (pH 6.9) overnight. Corneas were dissected out and stored at 48C in either phosphate buffered saline (PBS, 0.1 M, pH 7.4) for later use in whole-mount immunohistochemistry, or in PBS with 20% sucrose until cryostat sectioned. To assess changes in epithelial innervation patterns after cauterization, corneas from four of the eight rats per time point group were immunohistochemically labeled with antisera raised against Protein Gene Product-9.5 (PGP), a commonly used pan-neuronal marker. Corneas from two rats were processed whole, and those from the other two rats were sectioned parallel to the corneal surface at 25 mm. Tissue was washed in blocking solution (0.1 M PBS, 0.3% Triton X, 10% normal goat serum) overnight at 48C, then incubated 12– 18 h in rabbit PGP primary antibody (1:1000, Biogenesis, Sandown, NH; diluted in PBS with 0.3% Triton X, and 2% normal goat serum). Sections were rinsed in PBS, and incubated overnight with goat-anti-rabbit secondary antibodies conjugated to Cyanine-3.18 (Cy-3, 1:400 –600; Jackson Immuno Research, West Grove, PA; diluted in PBS). After being washed in PBS, sections were mounted on glass slides, dehydrated, cleared in xylene, and coverslipped using DPX (Fluka, Milwaukee, WI). Slides were first examined using conventional fluorescent microscopy. Selected sections were then imaged using single color laser scanning confocal microscopy. Corneas from the remaining four rats per group were processed using standard histological staining protocol and examined for evidence of corneal edema and immune cell infiltration, two common measures of inflammation. Corneas were sectioned at 5 mm perpendicular to the epithelial surface, and stained using Harris’s Hematoxylin and Eosin dyes (H&E, Surgipath Inc, Richmond, IL). Inflammatory edema was assessed by measuring thickness of the stroma, the dense collagen layer (also called
the substantia propria), which makes up the bulk of the cornea. The epithelium was not included in these measurements, as portions of it detached following cauterization. Stromal thickness was measured on cryostat sections using standard light microscopy with a calibrated eye piece. To control for possible differences in tissue section width caused by minor variations in sectioning angle, the cauterized and contralateral non-cauterized corneas from each rat were sectioned in the same cryostat chuck. The two corneas from each rat were stacked together and placed in a small plastic mold that was filled with mounting media and frozen by immersion in liquid nitrogen. The frozen block containing the corneas was removed from the mold and positioned on the cryostat chuck so that the epithelial surface of the corneas was perpendicular to plane of sectioning. This produced pairs of corneal cross-sections mounted side by side on each microscope slide, one section from the cauterized eye and the other from the contralateral eye of the same rat. Thickness of the cauterized cornea was then reported as relative thickness of the contralateral cornea at the adjacent site along the paired sections. Four pairs of sections per cornea were measured. Since the rat cornea is naturally thicker towards the periphery and thinner in the center, three measurements were taken at 150 mm intervals from the radial edge of the cornea and averaged. To determine the extent of immune cell infiltration, H&E stained sections were examined under high magnification. For each rat, identified immune cells were counted in four pairs of sections randomly chosen from those that contained the lesion site on the cauterized cornea. The mean number of cells per section was used to compare immune cell infiltration in the cauterized relative to the contralateral cornea for each rat, with no attempt made to extrapolate the density of cells in the whole cornea. To estimate the distribution of immune cells relative to the lesion site over time, the radial distance of identified immune cells from the edge of the cornea was recorded using morphological analysis software (Neurolucida, Microbrightfield, Burlington, VT). For each time point, three cauterized corneal sections were examined from each of the four rats. On each section, a point at half the corneal thickness at the junction of the cornea and sclera was selected and designated as the ‘origin point’. The coordinates of each tagged immune cell were recorded relative to that origin. X-coordinates were expressed as a ratio of the distance from the corneal periphery to the edge of the lesion. Distance ratios from the 12 sections per time point were pooled, and histograms generated using a graphics software package (SigmaPlot, SPSS, Chicago, IL). 2.3. Behavioral testing Eight separate groups of six rats each were tested for corneal sensitivity to an acute capsaicin stimulus. Each group was only tested once. The first group consisted of
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naı¨ve rats that had not been cauterized in either eye. Each of the other groups consisted of animals that had one cauterized and one healthy cornea. One group of six rats was tested at each of the following experimental time points: 2, 4, 6, 8, 12, 24 and 48 h after cauterization. Rats were mildly sedated with xylazine (0.5 ml/kg, IM), and behavioral testing was initiated when spontaneous movements ceased, but while pinching the paw with a pair of forceps elicited a brisk withdrawal reflex. The low dose of drug was not intended to anesthetize the rats, only to make them passive enough to allow testing without the use of restraint. Using a micropipetter, 4 ml of vehicle (PBS, 0.003% EtOH, 0.003% Tween-20) was applied to the eye being tested, followed by an equal amount of a 1 mM capsaicin solution. Extreme care was taken not to touch any part of the eye or surrounding hair and lashes. Since hyperalgesia is defined as an increased responsiveness to painful stimuli, the concentration of capsaicin was chosen based on results of preliminary experiments. The selected concentration of 1 mM consistently evoked a mild protective blinking response in non-inflamed eyes. Vehicle and capsaicin solutions were warmed to surface eye temperature (328C as measured with a contact thermocouple) in a water bath to control for a temperature response. Under these testing conditions rats did not blink spontaneously, allowing for easy identification of stimulus-induced blinking. Application of the capsaicin stimulus induced an immediate and discrete burst of rapid blinking that ended abruptly within 20 s. The response was scored by counting the number of blinks made in the 30 s time period following the application of the capsaicin eye drop. The eye was videotaped during testing. The tape was later played back in slow motion and the number of blinks recorded for each trial. One eye was tested at a time, and the order of testing (cauterized or control eye first) was alternated for each rat. Since capsaicin has been reported to cause desensitization at low concentrations, each eye was only tested once, and with a single dose of capsaicin. This also ensured that the acute capsaicin stimulus itself would not induce any further inflammatory reaction in the cornea and confound the results. Therefore a different group of animals was used at each of the eight time points examined, for a total of 48 rats tested. The use of a low dose of sedative did not appear to interfere with either eye blink or paw withdrawal reflex, and was necessary to allow accurate and reproducible testing behaviors. Non-sedated rats often exhibited other types of avoidance behaviors such as head shakes and paw wipes, which were not consistent from animal to animal. Also, it was important for the animals’ heads to remain relatively still during the duration of the testing, so that the blinking movement could be videotaped. Attempts to restrain the animals in a variety of both commercially available and custom-constructed restraint devices were unsuccessful, in
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part due to stress of immobilizing the head. Examination of videos played back in slow motion allowed accurate counting of rapid blinking movements. 2.4. Data analysis Two-way analyzes of variance (ANOVA), and Bonferroni multiple comparison procedure post hoc tests were used to compare data from normal corneas to those from cauterized corneas across the different time points. All data reported are expressed as group mean ^ standard deviation.
3. Results 3.1. Anatomical changes following chemical injury Histological changes were assessed by examination of the H&E stained cornea sections. Immediately following cauterization with silver nitrate, a discrete area of damage to the epithelial cell layer was evident as a yellowish brown patch. One hour later this patch was more pronounced, but there was no histological evidence of inflammation in the surrounding tissue (Fig. 1A). Four hours after cauterization, the damaged portion of the epithelial cell layer detached and edema of the underlying stroma was clearly evident. However, there was still little or no immune cell infiltration to the lesion site (Fig. 1B). At 6– 12 h, corneas became increasingly thickened, and immune cells were present in the wound area (Fig. 1C). By 24 h, immune cells were highly concentrated around the lesion site (Fig. 1D) and were still evident at 48 h. Immune cells were identified morphologically by examination of the H&E stained sections under high power, and consisted of approximately 92% neutrophils and 8% macrophages. Neutrophils are easily identified in H&E stained tissue by their multi-lobed nucleus and the grainy appearance of their cytoplasm, while macrophages are recognized by their large size and often irregular shape. No mast cells, lymphocytes or eosinophils were identified. At 1 week, three of the four corneas still demonstrated some signs of swelling and increased immune cell count in the area of the lesion. By 2 weeks, the inflammation had resolved, although in most cases, the regenerating corneal epithelium appeared thinner than that of the surrounding cornea. By 4 weeks, the cauterized and contralateral corneas were indistinguishable. PGP labeling showed a complete lack of terminal staining in the wound area 4 h after cauterization. This corresponds to the time point at which outer damaged epithelial cell layers detach, leaving a crater-like gap in the epithelium. This is clearly shown in Fig. 2. Within the wound boundary, bundles of sensory nerve axons running through the upper stroma are clearly visible due to loss of the outer epithelial layer.
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Fig. 1. Histological changes during development of corneal inflammation. (A– C) H&E stained cross-sections of silver nitrate cauterized (right) and contralateral non-cauterized (left) corneas at 1 h (A); 4 h (B); and 12 h (C) after chemical cauterization with silver nitrate. (D) Twenty-four hours after cauterization, immune cells accumulate in corneal stroma. S ¼ stroma; and E ¼ epithelium. Scale bar ¼ 100 mm in A; 75 mm in B and C; and 50 mm in D.
Fig. 2. Confocal micrograph of Protein Gene Product 9.5 immunoreactivity in the cornea 12 h after silver nitrate cauterization. At this time point, the cauterized section of epithelium has detached, taking with it the long parallel sensory fibers normally innervating the outer cell layers. Arrows point to edge of circular wound in central cornea. Due to removal of the epithelium, the upper layers of the stroma are exposed in the wound crater, revealing the sub-epithelial nerve plexus. Scale bar ¼ 200 mm. Inset (bottom left) depicts an outline drawing of whole mounted cornea. Corneas were processed free-floating, and radial cuts made so that the corneas could be flattened into a ‘pin-wheel’ shape and mounted on glass microscope slides. The filled gray circle in the center indicates area of silver nitrate lesion. The small rectangle indicates area of confocal micrograph.
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Fig. 3. Changes in corneal thickness with time. Edema begins within 4 h of cauterization. Maximum effect is seen by 24 h. Asteriks denote time points at which mean thickness for cauterized corneas is significantly greater than mean thickness for non-cauterized contralateral corneas. Asteriks also indicate that mean thickness for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:01; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
Fig. 4. Changes in immune cell count with time. Neutrophils begin to accumulate at the edge of the cauterized cornea within 4 h. Maximum effect was seen by 24 h. Asteriks denote time points at which mean sectional immune cell count for cauterized corneas is significantly greater than that of non-cauterized contralateral corneas. Asteriks also indicate that mean cell count for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:01; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
Beginning approximately 4 h after cauterization, silver nitrate treated corneas were significantly thicker than the non-cauterized contralateral corneas of the same rats (P , 0:01). Cauterized corneas were also significantly thicker than the corneas of naive control group rats (P , 0:01). No significant difference was found between the non-cauterized contralateral corneas of the experimental group rats, and the corneas of the naı¨ve control group animals, indicating that there were no centrally-mediated contralateral effects on corneal thickness. By 24 h after cauterization corneal thickness doubled, increasing from an average of 156 ^ 30 mm before cauterization to 309.2 ^ 21.6 mm. Thickness was still significantly elevated at 48 h (Fig. 3). Immune cell count, defined as the average number of histologically identified immune cells per 5 mm thick radial section of cornea, was extremely low for non-cauterized corneas, with an average count of 2.1 ^ 0.5 cells. No significant difference was found between the non-cauterized contralateral corneas and the corneas of the naive control group rats, again indicating the absence of a contralateral inflammatory effect. At 4 h, the mean immune cell count in the cauterized corneas became significantly elevated (P , 0:01), with an average of 65.1 ^ 7 cells per section. The count rose to 337.8 ^ 24 cells by 6 h, peaking at 24 h post-cauterization with a count of 479 ^ 125 cells per section. There was no significant difference between the 24 and 48 h groups (Fig. 4).
the scleral blood vessels at the edge of the cornea to reach the lesion in the central cornea. This results in a slower rate of immune cell accumulation than what is typically seen in cutaneous inflammatory reactions. The six histograms in Fig. 5 illustrate the migration of immune cells from the edge of the cornea (0%) to the edge of the lesion (100%). Two hours after cauterization, no immune cells were identified in the cornea. By 4 h, neutrophils were seen only around the periphery. Migration continued inward towards the center of the cornea, with cells reaching the edge of the lesion by 6 h after cauterization. The main area of concentration however, was still clearly confined to the periphery. Eight hours postcauterization, there was an almost even distribution of immune cells along the radial axis of the corneas. At 12 h, the distribution had shifted away from the peripheral edge of the cornea and towards the center, and by 24 h, immune cells, still consisting almost entirely of neutrophils, were clearly concentrated around the lesion in the middle of the cornea.
3.2. Time course of immune cell infiltration In addition to changes in the number of immune cells present in the cornea, we also sought to determine the time point at which immune cells reached the lesion site. Since the cornea is avascular, immune cells must migrate from
3.3. Assessment of corneal sensitivity Blinking response to a dilute capsaicin solution was significantly elevated (P , 0:05) 2 h post-cauterization, with an average of 31.3 ^ 8 blinks in the cauterized eye, compared to 17.5 ^ 5 blinks in the non-cauterized contralateral eye. By 6 h the hyperalgesia appeared to peak, with an average of 44.7 ^ 11.5 blinks in the cauterized eye as compared to 20 ^ 11 blinks in the contralateral eye. Responses returned to normal within 24 h after cauterization. No significant difference was observed between mean response in contralateral non-cauterized eyes at each time point, and mean response in either eye of the naı¨ve control group rats, indicating the absence of a centrally-mediated contralateral effect on corneal sensitivity (Fig. 6).
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Fig. 6. Changes in corneal sensitivity over time. Graph shows number of eye blinks in response to a dilute capsaicin solution applied to inflamed and contralateral corneas of rats 2, 4, 6, 8, 12, 24 and 48 h after corneal cauterization with silver nitrate. Increased response in the cauterized eye begins by 2 h, and resolves by 24. Maximum effect is seen 6 h after cauterization. Asteriks denote time points at which mean number of blinks for cauterized corneas is significantly greater than for non-cauterized contralateral corneas. Asteriks also indicate that mean number of blinks for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:05; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
inflammation then dropped quickly, returning to baseline by 24 h post-cauterization. In contrast, the two anatomical measures of inflammation (edema and immune cell count) remained elevated up to 48 h after silver nitrate treatment.
4. Discussion Chemical cauterization of the central cornea resulted in an acute inflammatory response as evidenced by the rapid development of edema and the infiltration of neutrophils. Fig. 5. Migration of immune cells towards the wound in the central cornea. Percent distance from the edge of the cornea to the chemically induced wound in the center is denoted along the x-axes, with 0% indicating the cornea-sclera border, and 100% indicating the edge of the lesion. Successive histograms demonstrate distribution of immune cells along the radial axis of the cornea at 2, 4, 6, 8, 12 and 24 h. Within 4 h, immune cell counts are elevated only in the periphery. Migration of immune cells then continues inwards towards the wound in the central cornea, reaching the edge of the wound at 6 h, then continuing to accumulate around the wound up to 24 h after cauterization.
3.4. Comparison of hyperalgesia and inflammatory time course Difference scores were computed for the cauterized versus contralateral eye for the thickness, immune cell count, and behavioral blink data from each rat. These scores were then expressed as a percent of the highest data value across the time points examined. This allowed all three measures to be graphed together so as to facilitate comparison of their time courses (Fig. 7). Hyperalgesia peaked more rapidly than the anatomical measures of
Fig. 7. Time course summary of corneal inflammation and hyperalgesia. For each of the three measures, thickness, immune cell count, and blink response, difference scores were computed for the cauterized versus noncauterized contralateral eyes of each rat. Scores were then expressed as percent of highest mean value across time points examined.
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Inflammation was accompanied by increased sensitivity to an acute noxious chemical stimulus to the eye, indicating the presence of corneal hyperalgesia. No significant differences were found between the contralateral non-cauterized corneas and the corneas of naı¨ve control group rats. The inner structures of the eye such as the retina are considered immune-privileged sites and usually do not exhibit inflammatory response except in the case of immune-compromised individuals. The cornea, on the other hand, is the outermost epithelial barrier of the eye, and as such, shares many characteristics with skin. Acute inflammation of the cornea resembles that of cutaneous tissue; both are characterized by edema, the infiltration of neutrophils, and increased sensitivity. Unlike skin however, the cornea is avascular. Thus in the central cornea, which is the furthest removed from limbal blood vessels, the onset of acute inflammation follows a slightly different time course. In all tissues, inflammation begins with an increase in vascular permeability and the subsequent movement of fluid, soluble plasma proteins, and immune cells from the blood vessels into the tissue. Within minutes to hours, this results in both edema and an accumulation of immune cells at the injury site. Neutrophils, also known as polymorphonuclear leukocytes, are the most numerous phagocytes found in the blood. The first immune cells to appear after tissue injury, neutrophils are a classic hallmark of acute inflammation. If inflammation persists and becomes chronic, other classes of immune cells begin to accumulate in the affected tissue, including lymphocytes, plasma cells and macrophages. In our model, over 90% of identified immune cells in the stroma were neutrophils. In the exterior segment of the eye, immune cells leaving the blood vessels at the periphery of the cornea must migrate through the stroma to reach a wound in the central cornea. Although edema occurs within an hour of tissue damage, immune cell accumulation at the wound site lags behind. In our model, elevated neutrophil counts in the cauterized cornea became noticeable in the periphery after 4 h, and in the central cornea after 6 h. Despite these differences, the development of inflammation in our model is in many ways consistent with models of cutaneous inflammation commonly used to study inflammatory sensitization, such as intraplantar injection of Complete Freund’s Adjuvant (CFA) or carrageenan. Care must be taken in comparing different models of inflammation, as the time course of any model is greatly influenced by the extent of injury or the amount of inflammatory agent used. However, certain aspects are common to all three models. For example, increased sensitivity usually resolves while anatomical inflammation is still in evidence, and peak edema and hyperalgesia may occur at distinct time points. In the present study, corneal sensitivity to capsaicin peaked at 6 h and resolved by 24 h, while edema and immune cell counts did not peak until 24 h and remained elevated 48 h after cauterization. Likewise, CFA-induced edema does not
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peak until approximately 5 days after injection, around the time that paw withdrawal latencies are returning to normal (Iadorola et al., 1988a; Hylden et al., 1988). This trend is also seen after intraplantar carrageenan injection, although carrageenan produces inflammation along a much shorter time course than does CFA. Thermal hyperalgesia develops rapidly, peaking between 1 and 4 h. Thermal sensitivity then returns to baseline within 12 h, while edema remains significantly elevated (Costello and Hargreaves, 1989; Joris et al., 1990). In all three models of persistent pain discussed above, the time courses of hyperalgesia and inflammation are distinct. The rapid onsets of enhanced tissue sensitivity suggest involvement of peripheral mechanisms. The return of behavioral measures to baseline while inflammation remains elevated may also be peripherally mediated. Alternatively, the relatively rapid resolution of hyperalgesia may involve efficient compensatory mechanisms within the central nervous system. When comparing different models, it is also important to consider the assay used to test for behavioral sensitization. Although rats exhibit increased responsiveness to both mechanical and thermal stimuli following intraplantar CFA injection, sensitization to heat often peaks well before sensitization to mechanical stimulation. Maximum decrease in thermal paw withdrawal has been reported at a wide range of time points, including 4– 6 h (Iadorola et al., 1988a; Hylden et al., 1988; Woolf et al., 1997), 8 h (Gould et al., 1999), and 24 h after injection (Iadorola et al., 1988b; Gould et al., 1998). On the other hand, maximum decrease in mechanical threshold may not occur until 24 –48 h after CFA injection (Iadorola et al., 1988a; Woolf et al., 1997). This difference in the time course of peak heat and mechanical sensitization, may be due to a number of centrally and peripherally mediated factors. One possibility is that different populations of primary afferent neurons mediate the behavioral responses to painful heat and mechanical stimuli, and that these populations may become sensitized independently of one another. In the cornea, capsaicin acts on polymodal primary afferent neurons that conduct in both the C and Ad ranges (Belmonte et al., 1991; Gallar et al., 1993). Since there is reported to be an overlap between responsiveness to capsaicin and noxious heat (Caterina et al., 1997; Tominaga et al., 1998), the same population of nociceptors may mediate behavioral response to both types of stimuli in the cornea. A separate population of corneal mechanoreceptors that conduct in the Ad range has been shown to be capsaicin-insensitive (Belmonte et al., 1991). It is therefore possible that a behavioral assay for corneal sensitivity to mechanical stimulation after silver nitrate induced inflammation would produce a different time course profile than did our assay for chemical sensitivity. This may be an interesting area for future study. It is likely that some aspects of silver nitrate induced corneal inflammation are neurogenic. The peripheral terminals of corneal primary afferent neurons contain neuropeptides such as vasoactive intestinal polypeptide, substance P
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and calcitonin gene-related peptide. These neuropeptides can be released antidromically during intense sensory stimulation, and contribute to the development of neurogenic inflammation by increasing capillary permeability and plasma extravasation (Gamse and Saria, 1985). Other models of chemically induced corneal irritation, such as the application of mustard oil, produce edema which can be blocked by preventing corneal nerve activation (Jampol et al., 1975; Camras and Bito, 1980). However, SP and CGRP alone do not appear to cause sensitization of corneal afferent neurons (Belmonte et al., 1994). Antidromic neuropeptide release by terminals at the border of the corneal lesion may therefore mediate the development of stromal edema, while sensitization of primary afferent neuronal response is mediated by other factors. An advantage to the corneal model is the degree to which the extent of the inflammatory reaction itself may be controlled. The spread of a subcutaneously injected inflammatory agent throughout the intra-plantar space is not completely uniform and boundaries between healthy and treated skin cannot readily be defined. Some researchers rely on multiple injections to the plantar surface as well as to individual toes to help insure even distribution of the agent and uniform effect across the entire plantar surface of the paw. With corneal cauterization, the extent and borders of the lesion are easily determined. The caustic properties of silver nitrate are neutralized by NaCl. A simple rinsing with saline therefore terminates the damaging action of the cauterizing agent. Since the lesion is produced by contact of the epithelium with a solid, the exact size, shape and location of the lesion can be delineated, and the extent of inflammation correlated with the distance from the lesion site. In this way we were able to determine the length of time necessary for immune cells to migrate through the stroma to the edge of the lesion in the central cornea. This model may be especially useful in examining immune cell migration during inflammatory conditions. Blinking of eyes is a complex behavior that can be initiated by both internal and extrinsic stimuli. It is mediated by multiple neural pathways and comprises sub-threshold neuromuscular events as well as nociceptive and nonnociceptive blinking behaviors. In the present study, we counted eye blinks in response to capsaicin as discrete events. In preliminary experiments, we determined that a range of capsaicin concentrations elicited eye blink responses that varied in a graded fashion. We chose to use a capsaicin concentration of 1 mM because it consistently produced small numbers of blinks in healthy corneas that were significantly greater than numbers of blinks observed following treatment with vehicle at the same temperature. Furthermore, the same concentration of capsaicin elicited a significantly greater number of blinks when applied to inflamed corneas. This concentration is also within the range of capsaicin concentrations reported to activate corneal nociceptors in cats (Belmonte et al., 1991; Gallar et al., 1993). Thus, we feel that within the range of stimulus
and drug concentrations used in the present experimental paradigm, the counting of eye blinks as all-or-none events provides a reliable and reproducible measure of a nociceptive behavior. There are many reasons why the cornea is well suited for the study of primary afferent nociceptors. Although there is some debate, pain appears to be the dominant sensation evoked by stimulation of the human cornea, suggesting that the majority of corneal sensory afferent neurons are nociceptors (Kenshalo, 1960; Beuerman and Tanelian, 1979; Lele and Weddell, 1956). However, cornea sensory cells represent a heterogeneous population with C and A-delta fiber units having been identified that respond to distinct stimulus modalities, including, polymodal nociceptors, cold receptors, as well as purely mechanoresponsive units. Thus the type and response properties of corneal primary afferent units appear to be fairly analogous to those present in glabrous skin, while offering advantages over skin such as increased simplicity of structure, greater innervation density, and more direct access to nerve terminals. Finally, a corneal model allows for the easy application of compounds without the need to inject them intradermally. This is advantageous for not only the administration of drugs, but also for the easy application of acute chemical stimuli such as capsaicin or acid. Accurate behavioral responses to the cutaneous injection of such agents can be problematic. We therefore believe that this model offers many advantages to currently available cutaneous models commonly used to study peripherally mediated pain.
Acknowledgements We thank Dr A.J. Beitz for careful reading and comment on an earlier version of this paper, and Samuel Roiko and Melodie Nannenga for technical assistance.
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H.N. Wenk, C.N. Honda / Pain 105 (2003) 393–401 Gallar J, Pozo MA, Tuckett RP, Belmonte C. Response of sensory units with unmyelinated fibres to mechanical, thermal and chemical stimulation of the cat’s cornea. J Physiol 1993;468:609– 22. Gamse R, Saria A. Potentiation of tachykinin-induced plasma protein extravasation by calcitonin gene-related peptide. Eur J Pharm 1985; 114(1):616. Gould III HJ, England JD, Liu ZP, Levinson SR. Rapid sodium channel augmentation in response to inflammation induced by complete Freund’s adjuvant. Brain Res 1998;802:69–74. Gould III HJ, Gould TN, Paul D, England JD, Liu ZP, Reeb S, Levinson SR. Development of inflammatory hypersensitivity and augmentation of sodium channels in rat dorsal root ganglia. Brain Res 1999;824: 296–9. Hylden JLK, Nahin RL, Traub RL, Dubner R. Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvantinduced inflammation: the contribution of dorsal horn mechanisms. Pain 1988;37:229– 43. Iadorola MJ, Douglass J, Civelli O, Naranjo JR. Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using CDNA hybridization. Brain Res 1988a;455:205–12. Iadorola MJ, Brady LS, Draisci G, Dubner R. Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity. Behavioral parameters and opioid receptor binding. Pain 1988b;35:313–26.
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Jampol LM, Neufeld AH, Sears ML. Pathways of the response of the eye to injury. Invest Ophthalmol Vis Sci 1975;14:84–189. Joris J, Costello A, Dubner R, Hargreaves KM. Opiates suppress carrageenan-induced edema and hyperthermia at doses that inhibit hyperalgesia. Pain 1990;43:95–103. Kenshalo DR. Comparison of thermal sensitivity if the forehead, lip, conjunctiva and cornea. J Appl Physiol 1960;15:987–91. Lele PP, Weddell G. Sensory nerves of the cornea and cutaneous sensibility. Exp Neurol 1956;1:334–59. Rozsa AJ, Beuerman RW. Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain 1982;14:105 –20. Sonoda K, Sakamoto T, Yoshikawa H, Ashizuka S, Ohshima Y, Kishihara K, Nomoto K, Ishibashi T, Inomata H. Inhibition of corneal inflammation by the topical use of ras farnesyltransferase inhibitors: selective inhibition of macrophage localization. Invest Ophthalmol Vis Sci 1998;39:2245 –51. Sunderkotter C, Beil W, Roth J, Sorg C. Cellular events associated with inflammatory angiogenesis in the mouse cornea. Am J Pathol 1991;138: 931– 9. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21:531–43. Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S. Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumor necrosis factor a. Br J Pharm 1997;121:417– 24.
Research paper
Silver nitrate cauterization: characterization of a new model of corneal inflammation and hyperalgesia in ratq H.N. Wenk, C.N. Honda* Department of Neuroscience and Graduate Program in Neuroscience, University of Minnesota, 6-145 Jackson Hall, 312 Church Street, Minneapolis, MN 55455-0329, USA Received 11 June 2002; received in revised form 27 August 2002; accepted 6 September 2002
Abstract Chemical cauterization of the central cornea with silver nitrate was assessed as a superficial injury model of tissue sensitization accompanying acute inflammation. Adult male Sprague– Dawley rats were anesthetized with halothane gas, and the centers of their right corneas treated with a silver nitrate applicator stick (75% silver nitrate, 25% potassium nitrate) to produce a discrete lesion 1 mm in diameter. Edema of the corneal stroma and elevated immune cell counts became significant 4 h after cauterization, and were still evident after 48 h. Behavioral sensitization to chemical stimuli was determined by counting the number of blinks following application of 1 mM capsaicin directly to the corneal surface. A significant increase in stimulus-induced blinking was evident 2 h after cauterization. Chemical sensitization peaked at 6 h, and was no longer significant at 12 h. We conclude that silver nitrate cauterization produces acute corneal inflammation and hyperalgesia, and may prove a useful model for the study of primary afferent nociceptors. q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Cornea; Inflammation; Hyperalgesia
1. Introduction The cornea is well suited for the study of primary afferent nociceptors. It is the most densely innervated epithelium in the body, with an innervation density up to 600 times that of skin, and up to 40 times that of tooth pulp (Rozsa and Beuerman, 1982). The cornea contains the same basic types of nociceptors as does skin, but in a simplified, avascular environment. In addition, free nerve endings terminate only a few microns below the corneal surface, allowing for more direct access to sensory nerve terminals and the ability to perform rapid and consistent delivery of topically administered compounds. Chemical cauterization of the murine central cornea by topical application of silver nitrate has been used to study ocular angiogenesis and injury-induced clouding of the cornea (Sunderkotter et al., 1991; Sonoda et al., 1998). However, this model has not been used in behavioral testing q
Supported by NIH grants DA09641 and DE07288. * Corresponding author. Tel.: þ 1-612-624-3915; fax: þ1-612-624-8118. E-mail address: [email protected] (C.N. Honda).
of ocular sensitivity or pain, and has not been used before in rat. The aim of this study is to develop and characterize a silver nitrate model of non-specific inflammation in the adult rat cornea, including behavioral assessment of accompanying changes in pain sensitivity.
2. Methods All experimental procedures were approved by the Institutional Animal Use and Care Committee at the University of Minnesota and conform to established guidelines. 2.1. Induction of inflammation Adult male Sprague – Dawley rats 250 – 300 g were deeply anaesthetized with halothane (4%, in air), and the centers of their right corneas cauterized with a silver nitrate applicator stick (75% silver nitrate, 25% potassium nitrate; Graham-Field Inc, Hauppauge, NY). The applicator was held in contact with the cornea for 2 s, producing a discrete
0304-3959/$30.00 q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0304-3959(03)00295-1
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grayish-white lesion 1 mm in diameter. The cauterized eye was then rinsed several times with room temperature saline, and a thin layer of ophthalmic ointment (Artificial Tears Ointment; lanolin, mineral oil, white petrolatum; Phoenix Pharmaceutical, St. Joseph, MO) was applied to both eyes. Rats recovered fully from anesthesia within a few minutes, and exhibited no outward signs of distress. A few rats kept the cauterized eye slightly closed for up to 4 h following the procedure. 2.2. Histology Both cauterized and contralateral non-cauterized corneas were collected from eight rats at each the following 11 time points after silver nitrate cauterization: 1, 2, 4, 6, 8, 12, 24, and 48 h, and 1, 2 and 4 weeks. In addition, eight naı¨ve rats were used as a control group. Rats were deeply anaesthetized with sodium pentobarbital (65 mg/kg, IP) and perfused through the heart with 0.9% saline at room temperature followed by 4% paraformaldehyde at 48C. Both eyes were removed and immersion-fixed in 4% paraformaldehyde with 0.4% picric acid (pH 6.9) overnight. Corneas were dissected out and stored at 48C in either phosphate buffered saline (PBS, 0.1 M, pH 7.4) for later use in whole-mount immunohistochemistry, or in PBS with 20% sucrose until cryostat sectioned. To assess changes in epithelial innervation patterns after cauterization, corneas from four of the eight rats per time point group were immunohistochemically labeled with antisera raised against Protein Gene Product-9.5 (PGP), a commonly used pan-neuronal marker. Corneas from two rats were processed whole, and those from the other two rats were sectioned parallel to the corneal surface at 25 mm. Tissue was washed in blocking solution (0.1 M PBS, 0.3% Triton X, 10% normal goat serum) overnight at 48C, then incubated 12– 18 h in rabbit PGP primary antibody (1:1000, Biogenesis, Sandown, NH; diluted in PBS with 0.3% Triton X, and 2% normal goat serum). Sections were rinsed in PBS, and incubated overnight with goat-anti-rabbit secondary antibodies conjugated to Cyanine-3.18 (Cy-3, 1:400 –600; Jackson Immuno Research, West Grove, PA; diluted in PBS). After being washed in PBS, sections were mounted on glass slides, dehydrated, cleared in xylene, and coverslipped using DPX (Fluka, Milwaukee, WI). Slides were first examined using conventional fluorescent microscopy. Selected sections were then imaged using single color laser scanning confocal microscopy. Corneas from the remaining four rats per group were processed using standard histological staining protocol and examined for evidence of corneal edema and immune cell infiltration, two common measures of inflammation. Corneas were sectioned at 5 mm perpendicular to the epithelial surface, and stained using Harris’s Hematoxylin and Eosin dyes (H&E, Surgipath Inc, Richmond, IL). Inflammatory edema was assessed by measuring thickness of the stroma, the dense collagen layer (also called
the substantia propria), which makes up the bulk of the cornea. The epithelium was not included in these measurements, as portions of it detached following cauterization. Stromal thickness was measured on cryostat sections using standard light microscopy with a calibrated eye piece. To control for possible differences in tissue section width caused by minor variations in sectioning angle, the cauterized and contralateral non-cauterized corneas from each rat were sectioned in the same cryostat chuck. The two corneas from each rat were stacked together and placed in a small plastic mold that was filled with mounting media and frozen by immersion in liquid nitrogen. The frozen block containing the corneas was removed from the mold and positioned on the cryostat chuck so that the epithelial surface of the corneas was perpendicular to plane of sectioning. This produced pairs of corneal cross-sections mounted side by side on each microscope slide, one section from the cauterized eye and the other from the contralateral eye of the same rat. Thickness of the cauterized cornea was then reported as relative thickness of the contralateral cornea at the adjacent site along the paired sections. Four pairs of sections per cornea were measured. Since the rat cornea is naturally thicker towards the periphery and thinner in the center, three measurements were taken at 150 mm intervals from the radial edge of the cornea and averaged. To determine the extent of immune cell infiltration, H&E stained sections were examined under high magnification. For each rat, identified immune cells were counted in four pairs of sections randomly chosen from those that contained the lesion site on the cauterized cornea. The mean number of cells per section was used to compare immune cell infiltration in the cauterized relative to the contralateral cornea for each rat, with no attempt made to extrapolate the density of cells in the whole cornea. To estimate the distribution of immune cells relative to the lesion site over time, the radial distance of identified immune cells from the edge of the cornea was recorded using morphological analysis software (Neurolucida, Microbrightfield, Burlington, VT). For each time point, three cauterized corneal sections were examined from each of the four rats. On each section, a point at half the corneal thickness at the junction of the cornea and sclera was selected and designated as the ‘origin point’. The coordinates of each tagged immune cell were recorded relative to that origin. X-coordinates were expressed as a ratio of the distance from the corneal periphery to the edge of the lesion. Distance ratios from the 12 sections per time point were pooled, and histograms generated using a graphics software package (SigmaPlot, SPSS, Chicago, IL). 2.3. Behavioral testing Eight separate groups of six rats each were tested for corneal sensitivity to an acute capsaicin stimulus. Each group was only tested once. The first group consisted of
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naı¨ve rats that had not been cauterized in either eye. Each of the other groups consisted of animals that had one cauterized and one healthy cornea. One group of six rats was tested at each of the following experimental time points: 2, 4, 6, 8, 12, 24 and 48 h after cauterization. Rats were mildly sedated with xylazine (0.5 ml/kg, IM), and behavioral testing was initiated when spontaneous movements ceased, but while pinching the paw with a pair of forceps elicited a brisk withdrawal reflex. The low dose of drug was not intended to anesthetize the rats, only to make them passive enough to allow testing without the use of restraint. Using a micropipetter, 4 ml of vehicle (PBS, 0.003% EtOH, 0.003% Tween-20) was applied to the eye being tested, followed by an equal amount of a 1 mM capsaicin solution. Extreme care was taken not to touch any part of the eye or surrounding hair and lashes. Since hyperalgesia is defined as an increased responsiveness to painful stimuli, the concentration of capsaicin was chosen based on results of preliminary experiments. The selected concentration of 1 mM consistently evoked a mild protective blinking response in non-inflamed eyes. Vehicle and capsaicin solutions were warmed to surface eye temperature (328C as measured with a contact thermocouple) in a water bath to control for a temperature response. Under these testing conditions rats did not blink spontaneously, allowing for easy identification of stimulus-induced blinking. Application of the capsaicin stimulus induced an immediate and discrete burst of rapid blinking that ended abruptly within 20 s. The response was scored by counting the number of blinks made in the 30 s time period following the application of the capsaicin eye drop. The eye was videotaped during testing. The tape was later played back in slow motion and the number of blinks recorded for each trial. One eye was tested at a time, and the order of testing (cauterized or control eye first) was alternated for each rat. Since capsaicin has been reported to cause desensitization at low concentrations, each eye was only tested once, and with a single dose of capsaicin. This also ensured that the acute capsaicin stimulus itself would not induce any further inflammatory reaction in the cornea and confound the results. Therefore a different group of animals was used at each of the eight time points examined, for a total of 48 rats tested. The use of a low dose of sedative did not appear to interfere with either eye blink or paw withdrawal reflex, and was necessary to allow accurate and reproducible testing behaviors. Non-sedated rats often exhibited other types of avoidance behaviors such as head shakes and paw wipes, which were not consistent from animal to animal. Also, it was important for the animals’ heads to remain relatively still during the duration of the testing, so that the blinking movement could be videotaped. Attempts to restrain the animals in a variety of both commercially available and custom-constructed restraint devices were unsuccessful, in
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part due to stress of immobilizing the head. Examination of videos played back in slow motion allowed accurate counting of rapid blinking movements. 2.4. Data analysis Two-way analyzes of variance (ANOVA), and Bonferroni multiple comparison procedure post hoc tests were used to compare data from normal corneas to those from cauterized corneas across the different time points. All data reported are expressed as group mean ^ standard deviation.
3. Results 3.1. Anatomical changes following chemical injury Histological changes were assessed by examination of the H&E stained cornea sections. Immediately following cauterization with silver nitrate, a discrete area of damage to the epithelial cell layer was evident as a yellowish brown patch. One hour later this patch was more pronounced, but there was no histological evidence of inflammation in the surrounding tissue (Fig. 1A). Four hours after cauterization, the damaged portion of the epithelial cell layer detached and edema of the underlying stroma was clearly evident. However, there was still little or no immune cell infiltration to the lesion site (Fig. 1B). At 6– 12 h, corneas became increasingly thickened, and immune cells were present in the wound area (Fig. 1C). By 24 h, immune cells were highly concentrated around the lesion site (Fig. 1D) and were still evident at 48 h. Immune cells were identified morphologically by examination of the H&E stained sections under high power, and consisted of approximately 92% neutrophils and 8% macrophages. Neutrophils are easily identified in H&E stained tissue by their multi-lobed nucleus and the grainy appearance of their cytoplasm, while macrophages are recognized by their large size and often irregular shape. No mast cells, lymphocytes or eosinophils were identified. At 1 week, three of the four corneas still demonstrated some signs of swelling and increased immune cell count in the area of the lesion. By 2 weeks, the inflammation had resolved, although in most cases, the regenerating corneal epithelium appeared thinner than that of the surrounding cornea. By 4 weeks, the cauterized and contralateral corneas were indistinguishable. PGP labeling showed a complete lack of terminal staining in the wound area 4 h after cauterization. This corresponds to the time point at which outer damaged epithelial cell layers detach, leaving a crater-like gap in the epithelium. This is clearly shown in Fig. 2. Within the wound boundary, bundles of sensory nerve axons running through the upper stroma are clearly visible due to loss of the outer epithelial layer.
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Fig. 1. Histological changes during development of corneal inflammation. (A– C) H&E stained cross-sections of silver nitrate cauterized (right) and contralateral non-cauterized (left) corneas at 1 h (A); 4 h (B); and 12 h (C) after chemical cauterization with silver nitrate. (D) Twenty-four hours after cauterization, immune cells accumulate in corneal stroma. S ¼ stroma; and E ¼ epithelium. Scale bar ¼ 100 mm in A; 75 mm in B and C; and 50 mm in D.
Fig. 2. Confocal micrograph of Protein Gene Product 9.5 immunoreactivity in the cornea 12 h after silver nitrate cauterization. At this time point, the cauterized section of epithelium has detached, taking with it the long parallel sensory fibers normally innervating the outer cell layers. Arrows point to edge of circular wound in central cornea. Due to removal of the epithelium, the upper layers of the stroma are exposed in the wound crater, revealing the sub-epithelial nerve plexus. Scale bar ¼ 200 mm. Inset (bottom left) depicts an outline drawing of whole mounted cornea. Corneas were processed free-floating, and radial cuts made so that the corneas could be flattened into a ‘pin-wheel’ shape and mounted on glass microscope slides. The filled gray circle in the center indicates area of silver nitrate lesion. The small rectangle indicates area of confocal micrograph.
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Fig. 3. Changes in corneal thickness with time. Edema begins within 4 h of cauterization. Maximum effect is seen by 24 h. Asteriks denote time points at which mean thickness for cauterized corneas is significantly greater than mean thickness for non-cauterized contralateral corneas. Asteriks also indicate that mean thickness for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:01; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
Fig. 4. Changes in immune cell count with time. Neutrophils begin to accumulate at the edge of the cauterized cornea within 4 h. Maximum effect was seen by 24 h. Asteriks denote time points at which mean sectional immune cell count for cauterized corneas is significantly greater than that of non-cauterized contralateral corneas. Asteriks also indicate that mean cell count for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:01; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
Beginning approximately 4 h after cauterization, silver nitrate treated corneas were significantly thicker than the non-cauterized contralateral corneas of the same rats (P , 0:01). Cauterized corneas were also significantly thicker than the corneas of naive control group rats (P , 0:01). No significant difference was found between the non-cauterized contralateral corneas of the experimental group rats, and the corneas of the naı¨ve control group animals, indicating that there were no centrally-mediated contralateral effects on corneal thickness. By 24 h after cauterization corneal thickness doubled, increasing from an average of 156 ^ 30 mm before cauterization to 309.2 ^ 21.6 mm. Thickness was still significantly elevated at 48 h (Fig. 3). Immune cell count, defined as the average number of histologically identified immune cells per 5 mm thick radial section of cornea, was extremely low for non-cauterized corneas, with an average count of 2.1 ^ 0.5 cells. No significant difference was found between the non-cauterized contralateral corneas and the corneas of the naive control group rats, again indicating the absence of a contralateral inflammatory effect. At 4 h, the mean immune cell count in the cauterized corneas became significantly elevated (P , 0:01), with an average of 65.1 ^ 7 cells per section. The count rose to 337.8 ^ 24 cells by 6 h, peaking at 24 h post-cauterization with a count of 479 ^ 125 cells per section. There was no significant difference between the 24 and 48 h groups (Fig. 4).
the scleral blood vessels at the edge of the cornea to reach the lesion in the central cornea. This results in a slower rate of immune cell accumulation than what is typically seen in cutaneous inflammatory reactions. The six histograms in Fig. 5 illustrate the migration of immune cells from the edge of the cornea (0%) to the edge of the lesion (100%). Two hours after cauterization, no immune cells were identified in the cornea. By 4 h, neutrophils were seen only around the periphery. Migration continued inward towards the center of the cornea, with cells reaching the edge of the lesion by 6 h after cauterization. The main area of concentration however, was still clearly confined to the periphery. Eight hours postcauterization, there was an almost even distribution of immune cells along the radial axis of the corneas. At 12 h, the distribution had shifted away from the peripheral edge of the cornea and towards the center, and by 24 h, immune cells, still consisting almost entirely of neutrophils, were clearly concentrated around the lesion in the middle of the cornea.
3.2. Time course of immune cell infiltration In addition to changes in the number of immune cells present in the cornea, we also sought to determine the time point at which immune cells reached the lesion site. Since the cornea is avascular, immune cells must migrate from
3.3. Assessment of corneal sensitivity Blinking response to a dilute capsaicin solution was significantly elevated (P , 0:05) 2 h post-cauterization, with an average of 31.3 ^ 8 blinks in the cauterized eye, compared to 17.5 ^ 5 blinks in the non-cauterized contralateral eye. By 6 h the hyperalgesia appeared to peak, with an average of 44.7 ^ 11.5 blinks in the cauterized eye as compared to 20 ^ 11 blinks in the contralateral eye. Responses returned to normal within 24 h after cauterization. No significant difference was observed between mean response in contralateral non-cauterized eyes at each time point, and mean response in either eye of the naı¨ve control group rats, indicating the absence of a centrally-mediated contralateral effect on corneal sensitivity (Fig. 6).
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Fig. 6. Changes in corneal sensitivity over time. Graph shows number of eye blinks in response to a dilute capsaicin solution applied to inflamed and contralateral corneas of rats 2, 4, 6, 8, 12, 24 and 48 h after corneal cauterization with silver nitrate. Increased response in the cauterized eye begins by 2 h, and resolves by 24. Maximum effect is seen 6 h after cauterization. Asteriks denote time points at which mean number of blinks for cauterized corneas is significantly greater than for non-cauterized contralateral corneas. Asteriks also indicate that mean number of blinks for cauterized corneas at that time point is significantly greater than that of the naı¨ve control group (C) rats. P , 0:05; two-way ANOVA with Bonferroni Multiple Comparisons post hoc tests.
inflammation then dropped quickly, returning to baseline by 24 h post-cauterization. In contrast, the two anatomical measures of inflammation (edema and immune cell count) remained elevated up to 48 h after silver nitrate treatment.
4. Discussion Chemical cauterization of the central cornea resulted in an acute inflammatory response as evidenced by the rapid development of edema and the infiltration of neutrophils. Fig. 5. Migration of immune cells towards the wound in the central cornea. Percent distance from the edge of the cornea to the chemically induced wound in the center is denoted along the x-axes, with 0% indicating the cornea-sclera border, and 100% indicating the edge of the lesion. Successive histograms demonstrate distribution of immune cells along the radial axis of the cornea at 2, 4, 6, 8, 12 and 24 h. Within 4 h, immune cell counts are elevated only in the periphery. Migration of immune cells then continues inwards towards the wound in the central cornea, reaching the edge of the wound at 6 h, then continuing to accumulate around the wound up to 24 h after cauterization.
3.4. Comparison of hyperalgesia and inflammatory time course Difference scores were computed for the cauterized versus contralateral eye for the thickness, immune cell count, and behavioral blink data from each rat. These scores were then expressed as a percent of the highest data value across the time points examined. This allowed all three measures to be graphed together so as to facilitate comparison of their time courses (Fig. 7). Hyperalgesia peaked more rapidly than the anatomical measures of
Fig. 7. Time course summary of corneal inflammation and hyperalgesia. For each of the three measures, thickness, immune cell count, and blink response, difference scores were computed for the cauterized versus noncauterized contralateral eyes of each rat. Scores were then expressed as percent of highest mean value across time points examined.
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Inflammation was accompanied by increased sensitivity to an acute noxious chemical stimulus to the eye, indicating the presence of corneal hyperalgesia. No significant differences were found between the contralateral non-cauterized corneas and the corneas of naı¨ve control group rats. The inner structures of the eye such as the retina are considered immune-privileged sites and usually do not exhibit inflammatory response except in the case of immune-compromised individuals. The cornea, on the other hand, is the outermost epithelial barrier of the eye, and as such, shares many characteristics with skin. Acute inflammation of the cornea resembles that of cutaneous tissue; both are characterized by edema, the infiltration of neutrophils, and increased sensitivity. Unlike skin however, the cornea is avascular. Thus in the central cornea, which is the furthest removed from limbal blood vessels, the onset of acute inflammation follows a slightly different time course. In all tissues, inflammation begins with an increase in vascular permeability and the subsequent movement of fluid, soluble plasma proteins, and immune cells from the blood vessels into the tissue. Within minutes to hours, this results in both edema and an accumulation of immune cells at the injury site. Neutrophils, also known as polymorphonuclear leukocytes, are the most numerous phagocytes found in the blood. The first immune cells to appear after tissue injury, neutrophils are a classic hallmark of acute inflammation. If inflammation persists and becomes chronic, other classes of immune cells begin to accumulate in the affected tissue, including lymphocytes, plasma cells and macrophages. In our model, over 90% of identified immune cells in the stroma were neutrophils. In the exterior segment of the eye, immune cells leaving the blood vessels at the periphery of the cornea must migrate through the stroma to reach a wound in the central cornea. Although edema occurs within an hour of tissue damage, immune cell accumulation at the wound site lags behind. In our model, elevated neutrophil counts in the cauterized cornea became noticeable in the periphery after 4 h, and in the central cornea after 6 h. Despite these differences, the development of inflammation in our model is in many ways consistent with models of cutaneous inflammation commonly used to study inflammatory sensitization, such as intraplantar injection of Complete Freund’s Adjuvant (CFA) or carrageenan. Care must be taken in comparing different models of inflammation, as the time course of any model is greatly influenced by the extent of injury or the amount of inflammatory agent used. However, certain aspects are common to all three models. For example, increased sensitivity usually resolves while anatomical inflammation is still in evidence, and peak edema and hyperalgesia may occur at distinct time points. In the present study, corneal sensitivity to capsaicin peaked at 6 h and resolved by 24 h, while edema and immune cell counts did not peak until 24 h and remained elevated 48 h after cauterization. Likewise, CFA-induced edema does not
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peak until approximately 5 days after injection, around the time that paw withdrawal latencies are returning to normal (Iadorola et al., 1988a; Hylden et al., 1988). This trend is also seen after intraplantar carrageenan injection, although carrageenan produces inflammation along a much shorter time course than does CFA. Thermal hyperalgesia develops rapidly, peaking between 1 and 4 h. Thermal sensitivity then returns to baseline within 12 h, while edema remains significantly elevated (Costello and Hargreaves, 1989; Joris et al., 1990). In all three models of persistent pain discussed above, the time courses of hyperalgesia and inflammation are distinct. The rapid onsets of enhanced tissue sensitivity suggest involvement of peripheral mechanisms. The return of behavioral measures to baseline while inflammation remains elevated may also be peripherally mediated. Alternatively, the relatively rapid resolution of hyperalgesia may involve efficient compensatory mechanisms within the central nervous system. When comparing different models, it is also important to consider the assay used to test for behavioral sensitization. Although rats exhibit increased responsiveness to both mechanical and thermal stimuli following intraplantar CFA injection, sensitization to heat often peaks well before sensitization to mechanical stimulation. Maximum decrease in thermal paw withdrawal has been reported at a wide range of time points, including 4– 6 h (Iadorola et al., 1988a; Hylden et al., 1988; Woolf et al., 1997), 8 h (Gould et al., 1999), and 24 h after injection (Iadorola et al., 1988b; Gould et al., 1998). On the other hand, maximum decrease in mechanical threshold may not occur until 24 –48 h after CFA injection (Iadorola et al., 1988a; Woolf et al., 1997). This difference in the time course of peak heat and mechanical sensitization, may be due to a number of centrally and peripherally mediated factors. One possibility is that different populations of primary afferent neurons mediate the behavioral responses to painful heat and mechanical stimuli, and that these populations may become sensitized independently of one another. In the cornea, capsaicin acts on polymodal primary afferent neurons that conduct in both the C and Ad ranges (Belmonte et al., 1991; Gallar et al., 1993). Since there is reported to be an overlap between responsiveness to capsaicin and noxious heat (Caterina et al., 1997; Tominaga et al., 1998), the same population of nociceptors may mediate behavioral response to both types of stimuli in the cornea. A separate population of corneal mechanoreceptors that conduct in the Ad range has been shown to be capsaicin-insensitive (Belmonte et al., 1991). It is therefore possible that a behavioral assay for corneal sensitivity to mechanical stimulation after silver nitrate induced inflammation would produce a different time course profile than did our assay for chemical sensitivity. This may be an interesting area for future study. It is likely that some aspects of silver nitrate induced corneal inflammation are neurogenic. The peripheral terminals of corneal primary afferent neurons contain neuropeptides such as vasoactive intestinal polypeptide, substance P
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and calcitonin gene-related peptide. These neuropeptides can be released antidromically during intense sensory stimulation, and contribute to the development of neurogenic inflammation by increasing capillary permeability and plasma extravasation (Gamse and Saria, 1985). Other models of chemically induced corneal irritation, such as the application of mustard oil, produce edema which can be blocked by preventing corneal nerve activation (Jampol et al., 1975; Camras and Bito, 1980). However, SP and CGRP alone do not appear to cause sensitization of corneal afferent neurons (Belmonte et al., 1994). Antidromic neuropeptide release by terminals at the border of the corneal lesion may therefore mediate the development of stromal edema, while sensitization of primary afferent neuronal response is mediated by other factors. An advantage to the corneal model is the degree to which the extent of the inflammatory reaction itself may be controlled. The spread of a subcutaneously injected inflammatory agent throughout the intra-plantar space is not completely uniform and boundaries between healthy and treated skin cannot readily be defined. Some researchers rely on multiple injections to the plantar surface as well as to individual toes to help insure even distribution of the agent and uniform effect across the entire plantar surface of the paw. With corneal cauterization, the extent and borders of the lesion are easily determined. The caustic properties of silver nitrate are neutralized by NaCl. A simple rinsing with saline therefore terminates the damaging action of the cauterizing agent. Since the lesion is produced by contact of the epithelium with a solid, the exact size, shape and location of the lesion can be delineated, and the extent of inflammation correlated with the distance from the lesion site. In this way we were able to determine the length of time necessary for immune cells to migrate through the stroma to the edge of the lesion in the central cornea. This model may be especially useful in examining immune cell migration during inflammatory conditions. Blinking of eyes is a complex behavior that can be initiated by both internal and extrinsic stimuli. It is mediated by multiple neural pathways and comprises sub-threshold neuromuscular events as well as nociceptive and nonnociceptive blinking behaviors. In the present study, we counted eye blinks in response to capsaicin as discrete events. In preliminary experiments, we determined that a range of capsaicin concentrations elicited eye blink responses that varied in a graded fashion. We chose to use a capsaicin concentration of 1 mM because it consistently produced small numbers of blinks in healthy corneas that were significantly greater than numbers of blinks observed following treatment with vehicle at the same temperature. Furthermore, the same concentration of capsaicin elicited a significantly greater number of blinks when applied to inflamed corneas. This concentration is also within the range of capsaicin concentrations reported to activate corneal nociceptors in cats (Belmonte et al., 1991; Gallar et al., 1993). Thus, we feel that within the range of stimulus
and drug concentrations used in the present experimental paradigm, the counting of eye blinks as all-or-none events provides a reliable and reproducible measure of a nociceptive behavior. There are many reasons why the cornea is well suited for the study of primary afferent nociceptors. Although there is some debate, pain appears to be the dominant sensation evoked by stimulation of the human cornea, suggesting that the majority of corneal sensory afferent neurons are nociceptors (Kenshalo, 1960; Beuerman and Tanelian, 1979; Lele and Weddell, 1956). However, cornea sensory cells represent a heterogeneous population with C and A-delta fiber units having been identified that respond to distinct stimulus modalities, including, polymodal nociceptors, cold receptors, as well as purely mechanoresponsive units. Thus the type and response properties of corneal primary afferent units appear to be fairly analogous to those present in glabrous skin, while offering advantages over skin such as increased simplicity of structure, greater innervation density, and more direct access to nerve terminals. Finally, a corneal model allows for the easy application of compounds without the need to inject them intradermally. This is advantageous for not only the administration of drugs, but also for the easy application of acute chemical stimuli such as capsaicin or acid. Accurate behavioral responses to the cutaneous injection of such agents can be problematic. We therefore believe that this model offers many advantages to currently available cutaneous models commonly used to study peripherally mediated pain.
Acknowledgements We thank Dr A.J. Beitz for careful reading and comment on an earlier version of this paper, and Samuel Roiko and Melodie Nannenga for technical assistance.
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