## 4 Discussion  
A mouse model of shrimp allergy was established in the present study by repeated application of tropomyosin to the skin. It was also shown that Raman microscopy can capture the changes in skin causing sensitization. These changes were characterized as relative reductions in the intensity of Raman bands at 399, 915, and 1073 cm\({}^{-1}\) on the outermost surface of the skin. The method of skin treatment might be important for making animal models of food allergy through epicutaneous sensitization. Both the frequency and method of skin sensitization have varied in previous studies. For example, [PERSON] et al. employed three 1-week ovalbumin exposures to tape-stripped skin [19], whereas [PERSON] et al. treated mice with 4% SDS for three exposures per week over a 2-week period [18]. As shown  
Figure 3: Use of Raman microscopy to detect changes in the skin with impaired barrier function. (**A**) Typical Raman spectra of the skin of a mouse subjected to tape-stripping and SDS application. (**B**) Box plots for the intensity of Raman bands at 399, 915, and 1073 cm\({}^{-1}\). Raman bands were obtained from 3 independent sites at a depth of 5, 10, and 15 \(\upmu\)m from the skin surface following tape-stripping and SDS application. The differences in intensity between treatments indicate that there is a change in the molecular species and structure that constitute the skin. * \(p\) < 0.05, by ANOVA followed by Bonferroni test. (**C**) Hematoxylin and eosin staining of dorsal skin. by our data, four rounds of epicutaneous sensitization with shrimp tropomyosin appeared to lead to desensitization, as reflected by the drop of specific IgE levels upon the fourth epicutaneous sensitization on day 21, as well as during the two intragastric challenges on days 28 and 35, suggesting that the allergen-loading dose and frequency of administration must be optimized to generate an anaphylactic reaction. As our model induced anaphylaxis with only three epicutaneous sensitizations, it could be useful in studies to elucidate allergic pathogenesis or to establish treatment methods. It would be preferable to establish a unified model of epicutaneous sensitization to enable the comparison of results from different studies. The skin consists of epidermis, dermis, and subcutaneous tissue layers, and the epidermis is composed of the stratum corneum and stratified epithelium. The stratum corneum constitutes the outermost epidermal barrier and consists of corneocytes, with intercellular lipids filling the spaces between cells. The inner layer of the stratum corneum, the stratified epithelium, consists of multiple layers of flattened keratinocytes with tight junctions and other barrier structures that restrict the movement of materials through the cell layer [20]. The stratum corneum, stratified epithelium, and dermis of mouse skin were evaluated in the present study at depths of 5, 10, and 15 um from the surface using confocal Raman microscopy [21]. These analyses suggested that changes in the stratum corneum are associated with susceptibility to sensitization. It is known that dendritic cells, which are normally located inside the tight junctions of stratified epithelium, extend their dendrites to below the stratum corneum to capture antigens when the stratum corneum is damaged [22], which connects to our result that only changes in the outermost layer of the skin were significant. The lack of findings of changes in the skin under optical microscopy suggests that some substances indicated by the Raman bands had little effect on the morphology of the cell cytoskeleton or degenerated deposits. The structure of the outermost surface of the skin is easily disrupted by surfactants and abrasives contained in cosmetics and detergents [23]. As there are few established treatments for adult food allergies, determining how to protect the skin from allergens is considered important.