The animal protocols used in this study were reviewed and approved based on the ethical and scientific care procedures of the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2013-0385). The 8-week-old IL-4/Luc/CNS-1 Tg mice used in this study were kindly provided by the National Institute of Food and Drug Safety Evaluation of the Korea Food and Drug Administration (Osong, Korea), while HR1 mice were purchased from Central Lab Animal Inc. (Seoul, Korea). All mice were provided with ad libitum access to a standard irradiated chow diet (Samtako Inc., Korea) and water for 15 days. IL-4/Luc/CNS-1 Tg mice were handled in the Pusan National University-Laboratory Animal Resources Center accredited by the Korea Food and Drug Administration (FDA) (Accredited Unit Number-000231) and AAALAC International according to the National Institutes of Health guidelines (Accredited Unit Number-001525).

Eight-week-old IL-4/Luc/CNS-1 Tg mice (n=10) produced by mating of IL-4/Luc/CNS-1 Tg mice and HR1 mice randomly divided into two groups. In the first group (AOO treated group, n=5), 100 µL of AOO was repeatedly spread on the dorsum of the ears daily for 2 weeks. In the second group (FA treated group, n=5), 100 µL of 4% FA solution in vehicle (4:1 AOO, v/v: AOO) was repeatedly spread on the dorsum of the ears daily for 2 weeks. After final application, animals in the subset were subject to bioluminescence imaging analysis and further study.

Large numbers of IL-4/Luc/CNS-1 Tg mice were produced by mating IL-4/Luc/CNS-1 Tg mice and HR1 mice as described in a previous study [9]. Founder mice containing the IL-4/Luc/CNS-1 transgene were identified by PCR of tail-derived genomic DNA. For DNA-PCR, 10 pmol each of sense (5'-CTC GCA TGC CAG AGA TCC TA-3') and antisense (5'-CCA CAA CCT TCG CTT CAA AA-3') primers were added into the genomic DNA template mixture, after which the reaction mixtures were subjected to 25 cycles of amplification. Amplification was conducted in a thermal cycler (Perkin-Elmer, Waltham, MA, USA) under the following conditions: 30 sec at 94℃, 30 sec at 62℃, and 45 sec at 72℃. The amplified PCR products were then separated by 1% agarose gel electrophoresis, after which the band patterns were detected using a Kodak Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY, USA).

After final treatment, five immune organs including the ALN, thymus, spleen, SLN and mesenteric lymph node (MLN) were collected from scarified mice, and their weights were determined using an electronic balance (Mettler Toledo, Greifensee, Switzerland). Additionally, ear thickness was measured to determine the degree of allergic skin inflammation induced by FA treatment using a thickness gauge (Digimatic Indicator, Matusutoyo Co., Tokyo, Japan).

In vivo imaging was conducted using an IVIS imaging system (Xenogen, Oakland, CA, USA) as previously described [10]. Briefly, IL-4/Luc/CNS-1 Tg mice were anesthetized with Zoletil and injected i.p. with 150 mg/kg of D-luciferin (Sigma-Aldrich, MO, USA). At 10 minutes after D-luciferin injection, images of mice were taken for 3 min using an IVIS imaging system and photons emitted from specific regions were quantified using the Living Image software (Xenogen). The in vivo luciferase activity was then expressed in photons per second. The intensity of luminescence was analyzed using the Living Imaging software (Xenogen).

The serum IgE concentration was measured using an ELISA kit (Shibayagi, Inc., Gunma, Japan) according to the manufacturer's instructions. Briefly, wells coated with antibody were washed with washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) three times, after which 50 µL of serum samples and standards diluted 20 fold with dilution solution were added to the wells and the plate was incubated for 2 h. Next, 50 µL of biotin-conjugated avidin was added after washing with the above solution and samples were incubated for 2 h. Horseradish peroxidase-conjugated detection antibodies were then diluted 5,000-fold with conjugate diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) and transferred to each well. The plates were subsequently incubated at room temperature for 1 h, after which they were washed with washing solution three times. An enzyme reaction was subsequently initiated by adding substrate solution and incubating the plate at room temperature in the dark for 20 min. Finally, the reaction was terminated by adding 2M H₂SO₄ solution and the absorbance at 450 nm was measured.

Ear skins were removed from IL-4/Luc/CNS-1 Tg mice, fixed in 10% formalin, embedded in paraffin wax, routinely processed, and then sectioned into 5 µm thick slices. Next, the skin sections were stained with hematoxylin & eosin (H&E), after which they were examined by light microscopy at 100× and 400× magnification for the presence of edema and inflammatory cell accumulation and to determine the cellular morphology. The thickness of the epidermis and dermis and the number of lipid pores and inflammatory cells were also measured using the Leica Application Suite (Leica Microsystems, Wetzlar, Germany).

Furthermore, the infiltration of mast cells into ear tissue was detected by staining with toluidine blue. After deparaffinization and dehydration, ear skin sections were stained with 0.25% Toluidine blue (Sigma-Aldrich) and examined by light microscopy for the presence of mast cells. The number of mast cells per specific area was measured with Leica Application Suite (Leica Microsystems) and viewed at 400× magnification.

RT-PCR was conducted to measure the relative quantities of luciferase mRNA in LN from IL-4/Luc/CNS-1 Tg mice. For RT-PCR analysis, LN were frozen in liquid nitrogen, then chopped with scissors and homogenized in RNAzol B solution (Tet-Test Inc., TX, USA). The isolated RNA was subsequently measured using a Biospec-nano spectrophotometer (Dong-il Shimadzu, Korea). Expression of the transgenes was assessed by RT-PCR using 5 µg of the total RNA from each tissue. Next, 500 ng of oligo-dT primer (Invitrogen, CA, USA) was annealed at 70℃ for 10 min. Complementary DNA, which was used as the template for further amplification, was synthesized by the addition of dATP, dCTP, dGTP, and dTTP with 200 units of reverse transcriptase. Subsequently, 10 pmol of the sense and antisense primers were added, and the reaction mixture was subjected to 32 cycles of amplification in a Perkin-Elmer Thermal Cycler as follows: 1 min at 94℃, 1 min at 62℃, and 1 min at 72℃. In each case, negative-RT controls were included to differentiate between DNA and RNA products. RT-PCR was also conducted using β-actin-specific primers to ensure RNA integrity. The primer sequences for mouse IL-4 were as follows: mIL-4, 5'-CAG TCG ATG TAC ACG TTC GTC AC-3' and 5'-CTC AGT ACT ACG AGT AAT CCA-3'. The sequences of the β-actin sense and antisense primers were 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3' and 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3', respectively. All samples were analyzed in triplicate, and the final PCR products were separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

LN collected from a subset of the groups (n=5 per group) was homogenized using a PRO-PREP™ Solution Kit (iNtRON Biotechnology, Sungnam, Korea) supplemented with half of a protein inhibitor cocktail tablet (Roche, Penzberg, Germany). After homogenization, samples were centrifuged at 10,000×g for 10 min. The prepared proteins were then electrophoresed through a 10% SDS-PAGE gel, after which they were transferred to a nitrocellulose membrane (Amersham Biosciences, Corston, UK) for 2 h at 45 V in transfer buffer (25 mM Trizma-base, 192 mM glycine, and 20% methanol). Efficiency of the transfer as well as equal protein loading was evaluated by staining the membrane with Amido Black Staining Solution (Sigma-Aldrich) and the gel with Coomassie Blue. Appropriate dilutions of primary antibodies, anti-VEGF antibody (Pepro Tech., Rocky Hill, NJ, USA), anti-IL-6 (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (Sigma-Aldrich) were added to the membranes and allowed to hybridize overnight at 4℃. After the antibodies were removed, the membrane was washed three times in a solution composed of 10 mM Trizma-base (pH 7.6), 150 mM NaCl, and 0.05% Tween-20 for 10 min. The membrane was subsequently incubated with horseradish peroxidase-conjugated anti-secondary antibody for 1 h at room temperature, after which it was washed again as described above and developed using an enhanced chemiluminescence detection system (Amersham Bioscience). The results were quantified using the Image Analyzer System (Eastman Kodak 2000MM, NY, USA) and expressed as the fold-increase over control values.

One-way ANOVA was used to identify significant differences between the AOO treated group and FA treated group (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). All values were expressed as the means±SD. A P value of <0.05 was considered significant.

To determine if repeated dermal exposure of low dose FA can induce the expression of IL-4, alterations in luciferase signals were measured throughout the body and in eight organs of IL-4/Luc/CNS-1 Tg mice using the Living Image software. Analysis of the whole body image revealed that the luciferase signal was only detected at high levels in the abdominal region of IL-4/Luc/CNS-1 Tg mice treated with FA, while it was not observed in the AOO treated group (Figure 1A). Moreover, analysis of the organ image revealed that higher luciferase signals only occurred in the SLN of IL-4/Luc/CNS-1 Tg mice treated with low dose FA relative to the AOO treated group (Figure 1B). Additionally, a significantly higher level of mouse IL-4 mRNA was detected in the SLN of mice treated with low dose FA (Figure 1C). Taken together, these results suggest that IL-4 expression was induced by repeated dermal exposure to low dose FA in the SLN of IL-4/Luc/CNS-1 Tg mice.

To characterize changes in ear phenotypes induced by repeated dermal exposure of low dose FA, the ear thickness and morphology were observed in IL-4/Luc/CNS-1 Tg mice over 2 weeks. The ear thickness rapidly increased in the FA treated group relative to the AOO treated groups (Figure 2C). Morphological analysis revealed that the outline of the ear vein became clear or thickened in the FA treated group relative to the AOO treated group, while ear color changed from a flesh tint to dark brown (Figure 2A, B). Therefore, these findings demonstrate that repeated dermal exposure to low dose FA could induce a severe increase in ear thickness and alteration of ear morphology.

To investigate whether low dose FA can induce an increase of immune-system functions, the weight of five organs associated with the immune system and serum IgE concentration were measured in IL-4/Luc/CNS-1 Tg mice. As shown in Table 1, exposure to low dose FA was found to induce an increase in ALN and thymus weight of IL-4/Luc/CNS-1 Tg mice, while the weight of the spleen, SLN and MLN were maintained. Furthermore, there was no difference in body weight between the AOO and FA treated groups.

Hyperproduction of IgE in blood serum is a characteristic of allergic hypersensitivity and an indicator of the magnitude of allergic immune response [11]. Therefore, the serum IgE concentration was measured in subset groups to determine if an increase in IgE concentration was induced by the repeated dermal exposure to low dose FA. A significant increase in serum IgE concentration was detected in FA treated IL-4/Luc/CNS-1 Tg mice relative to AOO treated mice (Figure 2D). Therefore, these results suggest that the exposure to low dose FA may contribute to enhancement of the ALN weight, thymus weight and IgE concentration in IL-4/Luc/CNS-1 Tg mice.

Changes in ear tissue histology in response to low dose FA were measured in the ear tissue of IL-4/Luc/CNS-1 Tg mice after exposure to low dose FA. The epidermis and dermis was thicker in the FA treated group than the AOO treated group, while lichenification was widely observed in the epidermal surface. Moreover, FA treatment resulted in a nearly 2-fold increase in adipocyte and inflammatory cells in the dermis layer relative to the AOO treated group (Figure 3A, B). Furthermore, it was well known that mast cells play an important role in asthma, eczema, itch, allergic rhinitis and allergic conjunctivitis [12]. Therefore, infiltration of mast cells in ear skin sections of FA treated IL-4/Luc/CNS-1 Tg mice were observed after staining with toluidine blue. The number of infiltrated mast cells was higher in the dermis of the FA treated group than the AOO treated group (Figure 3Ae, f, Be). Taken together, these results indicate that the exposure of low dose FA could induce histological changes in AD morphology, including an increase in the epidermis and dermis, enhancement of adipocytes and infiltration of inflammatory cells.

In addition, some of the chemokines and cytokines secreted from irritant sites could contribute to the regulatory mechanism responsible for differences in skin irritation following exposure to various concentration of PA [9,13]. Therefore, to determine if exposure to low dose FA was accompanied by the secretion of cytokines, the expression of IL-6 and VEGF was measured in ear tissue of IL-4/Luc/CNS-1 Tg mice. The expression pattern of the two cytokines differed greatly between groups. Specifically, the expression of IL-6 was about 2 times higher in the low dose FA treated group than the AOO treated group, while VEGF expression was maintained a constant level (Figure 4). Therefore, these results showed that exposure to low dose FA may stimulate increased IL-6 expression in the ear tissue of IL-4/Luc/CNS-1 Tg mice.