INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SKIN CONTACT SENSITIZERS ARE simple chemical compounds that trigger localized lymphocytic inflammation (16, 17, 26). Distinct from irritant inflammation that persists for only 24-48 h, skin contact sensitizers stimulate induration and erythema that can persist for a week or more (9, 13). The characteristic induration and erythema associated with skin contact sensitizers likely reflects important physiological processes necessary for the development of lymphocytic inflammation. The induration may reflect the enhanced lymphatic flow necessary for the delivery of antigen and antigen-bearing cells to the regional lymph node (8). The increase in regional blood flow may be necessary to meet the enhanced metabolic demands of the antigen-stimulated tissues (43) as well as increase the delivery of lymphocytes to the site of antigenic stimulation (42). Recent studies in lymphocyte trafficking suggest that the regulation of lymphocyte migration into the inflammatory tissue occurs at the level of the microcirculation (39). These observations suggest that the lymphatic and vascular contributions to lymphocytic inflammation reflect a coordinated and interdependent response.

Attempts to concurrently evaluate the blood and lymph flow stimulated by skin contact sensitizers have been limited by the interdependence of these processes. Direct visualization of the microcirculation (e.g., intravital microscopy or laser-Doppler flowmetry) can be inhibited by the thickened tissue present during the inflammatory reaction (40). The repeated measurement of vascular tracers (e.g., radioactive or fluorescence markers) in lymphocytic inflammation may be restricted because of extravascular background activity (41). The utility of other tracers (e.g., hydrogen gas or electromagnetic flowmeters) can be limited by the requirement for implantable detection probes (28).

In this report, we evaluated the induration and erythema associated with epicutaneous oxazolone. Lymph flow was continuously monitored with efferent lymph duct cannulations of the prescapular lymph node. Blood flow to the oxazolone-stimulated skin was noninvasively measured by using a portable spectrophotometer and was confirmed by vascular corrosion casting. The results suggest that oxazolone stimulates a biphasic response involving an initial phase of increased lymph flow correlated with increased skin thickness. The second phase is defined by an increase in blood flow associated with structural adaptations in the superficial vascular network.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. Randomly bred male sheep, ranging in weight from 25 to 35 kg, were used in these studies. Sheep were excluded from the analysis if there was any gross or microscopic evidence of dermatitis. The sheep were given free access to food and water. The care of the animals was consistent with guidelines of the American Association for Accreditation of Laboratory Animal Care (Bethesda, MD).

Ear thickness measurements. Measurements were obtained on a shaved ear 10-12 cm from the auriculocranial junction near the tip of the ear. To avoid the midline cartilaginous ridge, the measurements were taken 2 cm from the dorsal ridge on the flat portion of the ear. The measurement site was marked with a 1.5-cm ink circle; this site was used for all subsequent measurements. Ear thickness was measured three times on both the oxazolone-stimulated and contralateral control ears with an Ames engineering micrometer (Ames, Waltham, MA) gently applied parallel to the contact surface. Data points typically represent the mean of three measurements.

Hematoxylin and eosin histology. After euthanasia, the oxazolone-stimulated and control tissues were harvested and immediately processed in parallel by quick freezing. Quick- frozen tissue was sliced into 4 × 4 × 4-mm blocks, coated with optimum cutting temperature (OCT) embedding media (TissueTek, Elkhart, IL), and placed in 15-mm cryomolds. The cryomolds were placed in liquid nitrogen-cooled 2-methylbutane followed by immersion in liquid nitrogen. The tissue was stored at 86°C for <3 mo before processing. The tissue was cut into 6-µm sections, immediately fixed in methanol (Fisher Scientific, Fair Lawn, NJ), and air dried. The slides were stained in Harris hematoxylin (Harris Modified SH26-F00D; Fisher) for 2 min followed by sequential rinses, including a brief acid rinse. The slides were counterstained with Eosin Y (0.5% eosin, 50% ethanol; Fisher) for 20 s then rinsed in ethanol and xylene (Fisher) followed by mounting with Permount (Fisher).

Digital image acquisition. The tissue sections were imaged on a Nikon Optiphot-2 microscope equipped with an episcopic fluorescence attachment. The microscope was equipped with ×10 binocular eyepiece tubes and ×20 and ×60 plan apochromat objectives. The images were recorded by use of a DC120 CCD camera (Kodak, Rochester, NY) with 24-bit color and 1,280 × 960 picture resolution. The images were processed by the MDS 120 system software (Kodak) and recorded as digitized TIFF files. The archived images were processed by use of the MetaMorph Imaging System 4.0 software (Universal Imaging, Brandywine, PA).

Distance measurement. Distance measurements was performed with the use of digital images of the hematoxylin and eosin histological sections. The 24-bit color images were thresholded based on a red-green-blue color space model. After thresholding, a 200 × 200-µm grid overlay was used to define 25-µm intervals. After standard distance calibration, the MetaMorph Imaging System 4.0 caliper application was used to measure epidermal and dermal thickness. The data were logged into Microsoft Excel 2000 (Redmond, WA) by dynamic data exchange. Dermal thickness was the distance from the cartilage-dermal junction to the epidermal basement membrane. The epidermis was the distance from the basement membrane of the basal cell layer to the stratum corneum.

Lymph duct cannulation. The prescapular lymph node (19) was used for all efferent lymph duct cannulations (34, 39). After general endotracheal anesthesia and sterile surgical preparation, an incision was placed in the jugular furrow 5 cm cephalad to the suprasternal notch (18, 27). The efferent lymph duct was cannulated with a heparin-bonded polyurethane catheter (Solo-Cath, CBAS-C35; Setters Life Sciences, San Antonio, TX). The cannula was passed through a 5-cm subcutaneous tunnel and secured at the skin. The lymph was collected in 50-ml sterile centrifuge tubes (Falcon, Franklin Lakes, NJ). Each tube contained 200 IU of heparin, 2,000 IU of penicillin (Cellgro, Mediatech; Herndon, VA), and 2,000 µg of streptomycin (Cellgro).

Spectrophotometry measurements. The Minolta portable spectrophotometer (CM-508d, Minolta, Ramsey, NJ) was used for all erythema measurements (Fig. 1). The spectrophotometer light source was a pulsed xenon arc lamp with a 400- to 700-nm wavelength range at a 20-nm pitch. The light from the xenon arc source passed through an integrating sphere to evenly illuminate the specimen surface. The illumination aperture was 11 mm with an 8-mm measurement aperture (the light reflected from the specimen surface at an angle of 8° was collected for measurement). The spectrophotometer had a photometric range of 0-175% and was calibrated with a white calibration cap. Triplicate measurements were obtained with automatic averaging. The spectrophotometric data were downloaded into proprietary software via a RS-232 port (SpectraMagic, Minolta). The data were transferred into Microsoft Excel 2000 spreadsheets to facilitate further analysis.

View larger version (96K): [in this window] [in a new window]   Fig. 1.   A: schematic diagram of the sheep ear demonstrating the relationship of the superficial venous plexus (SVP) and the superficial arterial plexus (SAP) to the epidermis (Ep) and cartilage (Ct). The hemoglobin content in the superficial vascular plexus was assessed by reflectance spectrophotometry. Incident light (Io) was reflected (R) by the skin layers and recorded by a Minolta spectrophotometer. B and C: anatomy of the superficial vascular plexus was confirmed by electron microscopy. Scanning electron micrograph demonstrating the horizontal orientation of the superficial venous plexus with interconnecting vessels (B, bar = 100 µm; C, bar = 20 µm).

Erythemal intensity measurement. The quantification of skin erythema was based on CIE (Commission Internationale de l'Eclairage) tristimulus color values based on calibrated spectrometric reflectance data from the ear and calculated by the SpectraMagic software application (CyberChrome, Minolta). Briefly, the tristimulus values corresponded to the intensities of the reflected light from the ear in red-green-blue bands. The erythemal intensity was calculated based on both the L*a*b* and the Munsell color systems. The L*a*b* color system uses a three-dimensional coordinate system with an L*-axis (brightness) and two orthogonal axes a* (red-green) and b* (yellow-blue) representing chromaticity (31). The Munsell notation is a similar coordinate system in which color is quantified as hue, value (brightness), and chroma (saturation) (38).

Corrosion casting. After systemic heparinization with 750 U/kg intravenous heparin, the external auricular arteries were bilaterally cannulated and perfused with ~100 ml of 37°C saline followed by a 2.5% buffered glutaraldehyde solution (Sigma Chemical) at pH 7.40. The casts were made by perfusion of the ear arteries with 100 ml of a Mercox (SPI, West Chester, PA) diluted with 20% methyl methacrylate monomers (Aldrich Chemical, Milwaukee, WI). After complete polymerization, the ears were harvested and macerated in 5% potassium hydroxide followed by drying and mounting for scanning electron microscopy. The microvascular corrosion casts were imaged after coating with gold in argon atmosphere with a Philips ESEM XL30 scanning electron microscope.

Image analysis of corrosion casts. The digital images of the oxazolone-stimulated and contralateral control corrosion casts were obtained at a standard distance and backlighting by use of a DC120 CCD camera (Kodak). After standard area calibration, the images of the corrosion casts were thresholded, and area measurements were obtained by use of the Metamorph image analysis software (Universal Imaging). To correct for sheep size variation, relative hole area was calculated. Relative hole area is the ratio of the hole area to the total area of the cast
A relative hole area of 0 indicates that the cast has no holes apparent in a two-dimensional projection, whereas a relative hole area of 1 indicates that the area of the cast is mostly holes. Morphometry of the electron micrographs of the corrosion casts have been described in detail elsewhere (22-24).

Statistical analysis. The sample analysis was based on multiple comparisons of paired-data Student-Newman-Keuls or Mann-Whitney test for nonparametric analysis of variance. The significance level for the sample distribution was defined as P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Skin thickness. Skin thickness after the application of oxazolone was assessed with an engineering micrometer. Triplicate measures of the stimulated and contralateral control ears were performed at 24-h intervals. The significant increase in the thickness of the oxazolone-stimulated ear detected at 24 h (Student's t-test; P < 0.001) persisted for >5 days (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The change () in ear thickness of the oxazolone-stimulated () and the contralateral control () ear. Triplicate measurements of the antigen-stimulated and control ears were performed at 24-h intervals for 120 h. The combined measurements of 6 consecutive sheep are shown; the error bars reflect 1 SD.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Histological morphometry of the oxazolone-stimulated (solid bars) and the contralateral control (open bars) ear. At various intervals after antigen stimulation, the sheep ears were vertically sampled and processed in parallel for histological examination. Images of the histological sections were digitized and calibrated for distance measurements. By using a 200 × 200-µm image grid with 25-µm gradations, measurements were made of the epidermis (A) and dermis (B) at 25-µm intervals. Data show mean measurements of paired ears with error bars reflecting 1 SD.

Lymph flow. The increase in skin thickness correlated with increased output of lymph plasma from the lymph node draining the oxazolone-stimulated skin. Within 24 h of oxazolone application, the prescapular lymph node demonstrated a marked rise in lymph plasma output. The lymph output peaked between 24 and 48 h after stimulation (Fig. 4A), and the mean output was 3.1-fold (n = 6 sheep) the output of the contralateral lymph node (Fig. 4B). The increase in lymph flow preceded the increase in cell output from the lymph node by 24-48 h (Fig. 4A).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Flow of the efferent lymph draining the oxazolone-stimulated (A) and contralateral control (B) prescapular lymph nodes in a representative sheep. The epicutaneous antigen was applied at time 0. Serial collections were performed throughout the experimental period. Cells collected after 24 h were exclusively mononuclear cells by light microscopy and flow cytometry (40).

Blood flow. The erythema of the antigen-stimulated skin was assessed at 24-h intervals by use of a portable spectrophotometer (Fig. 5). The comparison of the oxazolone-stimulated and contralateral control ears showed a marked increase in erythema in the antigen-stimulated skin. By using both the CIE and Munsell color space models, erythema increased to a maximum at 72-96 h after oxazolone stimulation (Fig. 5). The erythema appeared to be the result of increased hemoglobin content, because a comparison of oxazolone-stimulated and control ears in six consecutive sheep demonstrated a marked difference in the reflectance spectrum of hemoglobin (500-670 nm) (Fig. 6). There was no significant change in the relative oxyhemoglobin and deoxyhemoglobin content from 0 to 120 h.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Combined spectrophotometric measurements of oxazolone-stimulated (solid line) and contralateral control (dashed line) ears in 6 sheep. The measurements were obtained at 0, 24, 48, 72, 96, and 120 h. The epicutaneously antigen was applied at time 0. Data points represent mean calculated value for erythema (a*; A) and erythemal intensity (C*; B) at each time point. Error bars reflect 1 SD.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Serial spectrophotometric measurements of oxazolone-stimulated (solid line) and contralateral control (dashed line) sheep ears. Mean measurements of 6 sheep are shown. Measurements were obtained at 0, 24, 48, 72, 96, and 120 h. Epicutaneously antigen was applied at time 0.

View larger version (125K): [in this window] [in a new window]   Fig. 7.   Corrosion casts of oxazolone-stimulated (A) and contralateral control (B) ears. Ears were flushed with heparinized saline and with the Mercox polymer 96 h after oxazolone stimulation. After tissue digestion, the digital images were acquired with controlled backlighting.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Digital morphometry of representative corrosion casts comparing the oxazolone-stimulated and control microcirculations. Vessel diameters were measured after standard distance calibration. The measurements of individual vessel segments in the oxazolone-stimulated () and control () are shown. Solid line shows mean microvessel diameter in the oxazolone corrosion cast. Dotted line shows mean diameter of the contralateral control circulation. Scattergram is shown using arbitrary vessel segment labels to facilitate presentation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this report, we evaluated the induration and erythema associated with exposure to the epicutaneous antigen oxazolone. Lymph flow was continuously monitored with efferent lymph duct cannulations of the draining prescapular lymph node. Blood flow to the oxazolone-stimulated skin was noninvasively measured by use of a portable spectrophotometer. The results suggest that oxazolone stimulates a biphasic response involving an initial phase of increased lymph flow correlated with increased skin thickness followed by an increased in blood flow associated with structural adaptations in the superficial vascular network.

Since the earliest vascular studies, the hallmark of acute inflammation has been the macroscopic changes produced by hyperemia. Cohnheim (10) originally described these changes as rubor and tumor, characteristics that he ascribed to enhanced blood flow and exudation. Subsequent work has suggested that rubor, or redness, is due to two events: 1) enhanced blood flow in the microcirculation due, at least initially, to relaxation of precapillary sphincters and the dilatation of small arterial vessels with a resulting engorgement of the microcirculation (20, 45) and 2) the extravasation of erythrocytes out of the microcirculation (25).

The basic findings of erythema and induration were confirmed by our studies. The spectrophotometric measurements confirmed the persistent erythema and the enhanced lymph flow was consistent with tissue induration. The time course of the erythema, however, suggested changes in the microcirculation beyond the dynamic vasoregulation observed in irritant inflammation (6, 40). Moreover, histological studies excluded red cell extravasation as a potential source of the ongoing erythema. To address the possibility of structural changes in the microcirculation, we used pressure-controlled corrosion casting to examine both the oxazolone-stimulated and control microcirculation. The corrosion casts, electron microscopy, and digital morphometry demonstrated significant dilatation of the antigen-stimulated microcirculation. These studies suggest that the persistence of the erythema may be due not only to active vasoregulation but also to structural adaptations by the inflammatory microvessels.

Previous work in autoimmune skin diseases have postulated a similar increase in venular diameter. Studying atopic skin diseases, detailed morphological studies by Braverman and colleagues have shown substantial venular proliferation and differential venular growth in antigen-stimulated skin (5, 7). Braverman and co-workers have also shown preferential uptake of tritiated thymidine by the proliferating endothelium (4, 6). Moreover, this change was reversible with effective treatment. The similar findings in clinical atopic skin diseases and experimental epicutaneous antigen suggests that microvascular dilatation may play a physiological role in lymphocytic inflammation. One potential function for these changes in the microcirculation is the delivery of lymphocytes to the region of inflammation. The skin contact sensitizer oxazolone produced erythema that persisted for 4 days. Interestingly, 96 h is the time frame for the peak of lymphocyte recruitment into the oxazolone-stimulated skin (39).

The portable spectrophotometer used in these studies appears to have a practical role in quantifying the extent of erythema in inflammatory reactions. Recent progress in optoelectronic engineering has resulted in a number of portable instruments that can reliably quantify skin color data (15, 33, 35-37). In the present experiments, we used a Minolta reflectance spectrophotometer (DM-508d) that was highly portable and provided reproducible reflectance measurements. The disadvantage of this system was the requirement for specialized software applications for data processing, the expense of the spectrophotometer, and the potential sampling error produced by the limited opening of the probe head (8 mm). Finally, the spectrophotometer provides a measure of hemoglobin content and not an assessment of flow velocity. The inference that increased hemoglobin content corresponds to increased blood flow is based on previous measures of cell velocity (40) and plasma flow (41) in this model.

The primary reason for using the reflectance spectrophotometer was its theoretical ability to provide a noninvasive longitudinal assessment of cutaneous blood flow. In normal circumstances, skin color is primarily determined by the scattering and absorption of incident light inside the skin. Dawson et al. (11) and Diffey et al. (12) have proposed a widely held model of these optical properties of skin. Briefly, the model defines four optical layers of the skin: the stratum corneum, the melanin-containing layer of the epidermis, the superficial vascular plexus in the upper dermis, and the dermis below. The uppermost layer is assumed to transmit most incident light whereas the dominant site of absorption occurs in the melanin- and hemoglobin-containing layers. The bottom layer is assumed to backscatter a large proportion of light irrespective of its wavelength.

There are several considerations in the application of this model to the measure of cutaneous blood flow in the sheep ear. First, the model assumes that diffuse reflection of light from the upper layers is largely irrelevant. Although this assumption has produced a reasonable quantification of erythema in real skin (11), it may be prone to error in the longitudinal assessment of edematous skin. Second, the model considers the deep layer as a "white plate." This assumption is one of the primary reasons to study the sheep ear, namely, a limited depth of dermis and a highly reflective cartilaginous barrier. Third, serial measurements were made in the same region of the ear to limit changes in melanin content and maximize sensitivity to changes in hemoglobin content.

The reflectance data for the inflamed skin have been converted into a variety of indexes that are designed to indicate the relative amounts of melanin, oxyhemoglobin, and deoxyhemoglobin (2, 3, 14, 21). Although these reflectance or quasi-absorbance values theoretically provide information about the amount of the chromophore in the skin (29, 32), the application of these indexes in acute lymphocytic inflammation is uncertain, and their reliable application will require further experimental confirmation. Despite the limitations of these indexes, no significant change in the ratio of oxyhemoglobin and deoxyhemoglobin was observed.

Regional lymph flow in these studies was monitored by using efferent lymph duct cannulations. The experimental advantage of efferent lymph duct cannulations was that 1) tissue lymph could be monitored remote from the site of antigen application, 2) the efferent duct provided an integrated sample of tissue lymph flow, and 3) and efferent duct cannulation avoided the local tissue trauma associated with so-called "pseudoafferents" (44). The disadvantage of cannulating the efferent lymph duct, in contrast to the afferent duct, is the potential for modification of the lymph plasma by the intervening lymph node. Previous work has shown that the efferent lymph can contain higher concentrations of protein than the afferent lymph (30). This effect appears to be the result of a "passive" process of water reabsorption consistent with the Starling principles of fluid exchange (1). In the present experiments, we suspect that passive modification of the lymph plasma may have contributed to variations in the magnitude of the response but is unlikely to have significantly altered the observed kinetics of the oxazolone-induced lymph flow.