- Email: [email protected]
Epicutaneous detection of transepidermally eliminated collagen by multiphoton microscopy: A possible non-invasive diagnosis method for acquired reactive perforating dermatosis
158
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
Letter to the Editor Epicutaneous detection of transepidermally eliminated collagen by multiphoton microscopy: A possible non-invasive diagnosis method for acquired reactive perforating dermatosis
Keywords: Acquired reactive perforating dermatosis; Multiphoton microscopy; Second harmonic generation; Human skin
Acquired reactive perforating dermatosis (ARPD) is an uncommon skin disorder characterized by transepidermal elimination of degenerated collagen fibers and elastic fibers [1]. Prurigo nodularis (PN) is quite often a differential diagnosis of ARPD. Histopathological detection of altered collagen fibers in the epidermis is essential for the diagnosis of ARPD. Skin biopsy is, however, invasive and sometimes requires serial sectioning. Multiphoton microscopy (MPM) is a useful tool for the non-invasive imaging of biological tissues such as collagen fibers of elastic fibers without exogenous probes [2]. Herein, we assessed whether MPM is applicable for the detection of transepidermal elimination of degenerated collagen fibers in ARPD.
Fig. 1. Emission of SHG light from transepidermally eliminated collagen fibers and degenerated collagen fibers in ARPD. (a, b, d, e) Multiphoton microscopy (MPM) imaging of deparaffinized samples of acquired reactive perforating dermatosis (ARPD) (a, b) and prurigo nodularis (PN) (d, e). Transepidermal elimination and degenerated collagen fibers showing second harmonic generation (SHG) light (blue signal) in the epidermis as a fiber or spots among the epidermis or crusts (a, b, arrowheads). No SHG signal in the epidermis or crust of PN (d, e) was detected. (c, f) Elastica van Gieson (EVG) staining of ARPD (c) and PN (f). (g, h) Clinical features of ARPD patient. Umbilicated papules (g) with the Koebner phenomenon (h). (i) EVG staining of the biopsy specimen revealed transepidermal elimination of degenerated collagen fibers among the epidermis and the crust (arrowheads). (j–l) MPM images of a vertically sectioned fresh sample of ARPD. SHG of collagen fibers detected among the crust (j, arrowhead), which was connected with collagen fibers in the papillary dermis. Autofluorescence of elastic fibers (k, arrowheads). Transepidermally eliminated collagen fibers in ARPD (c, arrowheads). SHG, second harmonic generation; AF, autofluorescence; C, crust; E, epidermis; D, dermis. Scale bars: 100 mm.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
A 76-year-old Japanese man with typical clinical findings of ARPD and three Japanese men in their seventies or eighties with PN were included in this study with written informed consent. The samples were taken using a biopsy punch and were cut into halves for histopathological evaluation and MPM imaging. Archived paraffin-embedded tissue samples from five patients of ARPD were also utilized for histopathological and MPM analysis as previously described [3]. MPM imaging was achieved with an excitation wavelength of 890 nm. Collagen fibers were detected by second harmonic generation (SHG) and autofluorescence through 420– 460 and 495–540 nm filters, respectively. In contrast, elastic fibers emitted only autofluorescence, which was detected solely through the latter filter. In the paraffin-embedded skin samples from the ARPD patient, we detected well-preserved SHG from dermal collagen fibers (Fig. 1a). Intriguingly, SHG was also detected in the epidermis and the crust (Fig. 1b, arrowheads). The distribution of SHG was similar to that of transepidermal elimination of degenerated collagen fibers observed with Elastica van Gieson (EVG) staining (Fig. 1c, arrowheads). In all the samples from four additional ARPD patients, SHG signal was detected in the epidermis and the crust in the same manner (Supplemental Fig. S1). No SHG was detected in the crust or epidermis of paraffin-embedded skin samples from PN patients (Fig. 1d and e) or healthy controls (data not shown), suggesting that SHG in the epidermis was a specific finding in ARPD.
159
Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.08.004. Next, in order to assess whether MPM detects SHG in a freshly excised sample, MPM observation was applied to the fresh skin biopsy sample from another patient with typical manifestations of ARPD (Fig. 1g and h). Umbilicated papules with severe pruritus distributed to the extremities and the body trunk (Fig. 1g) accompanied with the Koebner phenomenon (Fig. 1h). A papule with an extruding crust on the back was biopsied and transepidermal elimination of degenerated collagen fibers was detected with EVG (Fig. 1i), which confirmed the diagnosis of ARPD. MPM analysis of a vertical section of the sample revealed SHG in the crust (Fig. 1j), and the SHG signal was connected to that of collagen fibers in the papillary dermis (Fig. 1j, arrowhead). Autofluorescence of elastic fibers was also observed in a similar distribution to SHG (Fig. 1k, arrowheads), which was consistent with the previous report of the simultaneous elimination of collagen and elastic fibers in ARPD [1]. Thus, MPM could visualize the transepidermal elimination of degenerated collagen fibers even in a fresh sample. Finally, to investigate the possibility of MPM as a non-invasive diagnostic tool for ARPD, freshly biopsied samples were observed from the skin surface side. As expected, SHG from collagen fibers was clearly detected in the crust of ARPD (Fig. 2b, arrowhead), whereas SHG was not observed in the crusts or epidermis of three samples of PN patients (Fig. 2e). These results indicated that MPM
Fig. 2. Detection of SHG light of collagen by MPM from epidermal side in ARPD and not in PN. MPM images of the epidermal side of a fresh sample of ARPD (a–d) and PN (e–g). (a) A low-magnification view of ARPD revealed a crust containing SHG of collagen fibers (square), which was clearly distinct from the normal epidermis surrounding the crust (arrowheads). (b–d) A high-magnification view of the crust (a, square) revealed collagen SHG (b, arrowhead) and AF of elastic fibers or keratinocytes (c, arrowhead). (e–g) A low-magnification view of PN. (e) No SHG signal was detected from the epidermal side of the PN sample. (f) Only autofluorescence of keratinocytes were detected. SHG, second harmonic generation; AF, autofluorescence. Scale bars: 500 mm.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
160
could detect transepidermally eliminated collagen fibers in ARPD even from the skin surface. MPM has several advantages for the observation of biological tissues. First, multiphoton laser excites fluorophores only within the focal point, thus the subcellular resolution is achieved even in highly scattering tissues. Second, the longer wavelength of the laser enables imaging of deep tissues with low photo-toxicity. Third, some biological components, such as elastic fibers, hair shafts, and keratinocytes, are detectable without exogenous probes because of their intrinsic fluorophores [2]. Moreover, collagen fibers can be visualized with SHG [2]. With these characteristics, MPM has been applied for the analysis of cell dynamics and structures of various organs, including skin, mainly in experimental animals [4–6]. However, recent reports suggest the possibility of MPM as a noninvasive diagnostic tool for human skin diseases, such as skin tumors, skin aging, and connective tissue diseases [2,3,7,8]. Compared to conventional histological analysis, a non-invasive diagnosis method offers two distinct benefits, namely, multi-lesion and chronological observation. Indeed, it is known that histologic findings in ARPD vary significantly even among the lesions of the same patient depending on their developmental stage [1]. Therefore, a non-invasive MPM analysis of multi-lesions would make it easier for dermatologists to find the degenerated collagen fibers in the epidermis of ARPD. Although it may be difficult to distinguish ARPD from other disorders of transepidermal elimination, such as perforating granuloma annulare by MPM imaging at present, detailed observations of these lesions might reveal the difference between them. Furthermore, a chronological observation of the same eruption may clarify the pathogenesis of ARPD in the future. Although limited to a single case, our results suggest the future possibility of MPM application for the non-invasive diagnosis of skin diseases with connective tissue degeneration, such as ARPD. Funding sources None.
References [1] Rapini RP, Herbert AA, Drucker CR. Acquired perforating dermatosis. Evidence for combined transepidermal elimination of both collagen and elastic fibers. Arch Dermatol 1989;125:1074–8. [2] Tsai TH, Jee SH, Dong CY, Lin SJ. Multiphoton microscopy in dermatological imaging. J Dermatol Sci 2009;56:1–8. [3] Murata T, Honda T, Miyachi Y, Kabashima K. Morphological character of pseudoxanthoma elasticum observed by multiphoton microscopy. J Dermatol Sci 2013;72:199–201. [4] Egawa G, Nakamizo S, Natsuaki Y, Doi H, Miyachi Y, Kabashima K. Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep 2013;3:1932. [5] Egawa G, Natsuaki Y, Miyachi Y, Kabashima K. Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy. J Dermatol Sci 2013;70:143–5. [6] Kabashima K, Egawa G. Intravital multiphoton imaging of cutaneous immune responses. J Invest Dermatol 2014. [7] Koehler MJ, Hahn S, Preller A, Elsner P, Ziemer M, Bauer A, et al. Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network. Exp Dermatol 2008;17:519–23. [8] Tong PL, Qin J, Cooper CL, Lowe PM, Murrell DF, Kossard S, et al. A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy. Br J Dermatol 2013;169:869–79.
Teruasa Murataa,b, Tetsuya Hondaa, Gyohei Egawaa, Yoshiki Miyachia, Kenji Kabashimaa,* a Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; bResearch Fellow of the Japan Society for the Promotion of Science, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3310; fax: +81 75 761 3002 E-mail address: [email protected] (K. Kabashima). Received 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.08.004
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
Letter to the Editor Epicutaneous detection of transepidermally eliminated collagen by multiphoton microscopy: A possible non-invasive diagnosis method for acquired reactive perforating dermatosis
Keywords: Acquired reactive perforating dermatosis; Multiphoton microscopy; Second harmonic generation; Human skin
Acquired reactive perforating dermatosis (ARPD) is an uncommon skin disorder characterized by transepidermal elimination of degenerated collagen fibers and elastic fibers [1]. Prurigo nodularis (PN) is quite often a differential diagnosis of ARPD. Histopathological detection of altered collagen fibers in the epidermis is essential for the diagnosis of ARPD. Skin biopsy is, however, invasive and sometimes requires serial sectioning. Multiphoton microscopy (MPM) is a useful tool for the non-invasive imaging of biological tissues such as collagen fibers of elastic fibers without exogenous probes [2]. Herein, we assessed whether MPM is applicable for the detection of transepidermal elimination of degenerated collagen fibers in ARPD.
Fig. 1. Emission of SHG light from transepidermally eliminated collagen fibers and degenerated collagen fibers in ARPD. (a, b, d, e) Multiphoton microscopy (MPM) imaging of deparaffinized samples of acquired reactive perforating dermatosis (ARPD) (a, b) and prurigo nodularis (PN) (d, e). Transepidermal elimination and degenerated collagen fibers showing second harmonic generation (SHG) light (blue signal) in the epidermis as a fiber or spots among the epidermis or crusts (a, b, arrowheads). No SHG signal in the epidermis or crust of PN (d, e) was detected. (c, f) Elastica van Gieson (EVG) staining of ARPD (c) and PN (f). (g, h) Clinical features of ARPD patient. Umbilicated papules (g) with the Koebner phenomenon (h). (i) EVG staining of the biopsy specimen revealed transepidermal elimination of degenerated collagen fibers among the epidermis and the crust (arrowheads). (j–l) MPM images of a vertically sectioned fresh sample of ARPD. SHG of collagen fibers detected among the crust (j, arrowhead), which was connected with collagen fibers in the papillary dermis. Autofluorescence of elastic fibers (k, arrowheads). Transepidermally eliminated collagen fibers in ARPD (c, arrowheads). SHG, second harmonic generation; AF, autofluorescence; C, crust; E, epidermis; D, dermis. Scale bars: 100 mm.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
A 76-year-old Japanese man with typical clinical findings of ARPD and three Japanese men in their seventies or eighties with PN were included in this study with written informed consent. The samples were taken using a biopsy punch and were cut into halves for histopathological evaluation and MPM imaging. Archived paraffin-embedded tissue samples from five patients of ARPD were also utilized for histopathological and MPM analysis as previously described [3]. MPM imaging was achieved with an excitation wavelength of 890 nm. Collagen fibers were detected by second harmonic generation (SHG) and autofluorescence through 420– 460 and 495–540 nm filters, respectively. In contrast, elastic fibers emitted only autofluorescence, which was detected solely through the latter filter. In the paraffin-embedded skin samples from the ARPD patient, we detected well-preserved SHG from dermal collagen fibers (Fig. 1a). Intriguingly, SHG was also detected in the epidermis and the crust (Fig. 1b, arrowheads). The distribution of SHG was similar to that of transepidermal elimination of degenerated collagen fibers observed with Elastica van Gieson (EVG) staining (Fig. 1c, arrowheads). In all the samples from four additional ARPD patients, SHG signal was detected in the epidermis and the crust in the same manner (Supplemental Fig. S1). No SHG was detected in the crust or epidermis of paraffin-embedded skin samples from PN patients (Fig. 1d and e) or healthy controls (data not shown), suggesting that SHG in the epidermis was a specific finding in ARPD.
159
Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.08.004. Next, in order to assess whether MPM detects SHG in a freshly excised sample, MPM observation was applied to the fresh skin biopsy sample from another patient with typical manifestations of ARPD (Fig. 1g and h). Umbilicated papules with severe pruritus distributed to the extremities and the body trunk (Fig. 1g) accompanied with the Koebner phenomenon (Fig. 1h). A papule with an extruding crust on the back was biopsied and transepidermal elimination of degenerated collagen fibers was detected with EVG (Fig. 1i), which confirmed the diagnosis of ARPD. MPM analysis of a vertical section of the sample revealed SHG in the crust (Fig. 1j), and the SHG signal was connected to that of collagen fibers in the papillary dermis (Fig. 1j, arrowhead). Autofluorescence of elastic fibers was also observed in a similar distribution to SHG (Fig. 1k, arrowheads), which was consistent with the previous report of the simultaneous elimination of collagen and elastic fibers in ARPD [1]. Thus, MPM could visualize the transepidermal elimination of degenerated collagen fibers even in a fresh sample. Finally, to investigate the possibility of MPM as a non-invasive diagnostic tool for ARPD, freshly biopsied samples were observed from the skin surface side. As expected, SHG from collagen fibers was clearly detected in the crust of ARPD (Fig. 2b, arrowhead), whereas SHG was not observed in the crusts or epidermis of three samples of PN patients (Fig. 2e). These results indicated that MPM
Fig. 2. Detection of SHG light of collagen by MPM from epidermal side in ARPD and not in PN. MPM images of the epidermal side of a fresh sample of ARPD (a–d) and PN (e–g). (a) A low-magnification view of ARPD revealed a crust containing SHG of collagen fibers (square), which was clearly distinct from the normal epidermis surrounding the crust (arrowheads). (b–d) A high-magnification view of the crust (a, square) revealed collagen SHG (b, arrowhead) and AF of elastic fibers or keratinocytes (c, arrowhead). (e–g) A low-magnification view of PN. (e) No SHG signal was detected from the epidermal side of the PN sample. (f) Only autofluorescence of keratinocytes were detected. SHG, second harmonic generation; AF, autofluorescence. Scale bars: 500 mm.
Letters to the Editor / Journal of Dermatological Science 76 (2014) 149–160
160
could detect transepidermally eliminated collagen fibers in ARPD even from the skin surface. MPM has several advantages for the observation of biological tissues. First, multiphoton laser excites fluorophores only within the focal point, thus the subcellular resolution is achieved even in highly scattering tissues. Second, the longer wavelength of the laser enables imaging of deep tissues with low photo-toxicity. Third, some biological components, such as elastic fibers, hair shafts, and keratinocytes, are detectable without exogenous probes because of their intrinsic fluorophores [2]. Moreover, collagen fibers can be visualized with SHG [2]. With these characteristics, MPM has been applied for the analysis of cell dynamics and structures of various organs, including skin, mainly in experimental animals [4–6]. However, recent reports suggest the possibility of MPM as a noninvasive diagnostic tool for human skin diseases, such as skin tumors, skin aging, and connective tissue diseases [2,3,7,8]. Compared to conventional histological analysis, a non-invasive diagnosis method offers two distinct benefits, namely, multi-lesion and chronological observation. Indeed, it is known that histologic findings in ARPD vary significantly even among the lesions of the same patient depending on their developmental stage [1]. Therefore, a non-invasive MPM analysis of multi-lesions would make it easier for dermatologists to find the degenerated collagen fibers in the epidermis of ARPD. Although it may be difficult to distinguish ARPD from other disorders of transepidermal elimination, such as perforating granuloma annulare by MPM imaging at present, detailed observations of these lesions might reveal the difference between them. Furthermore, a chronological observation of the same eruption may clarify the pathogenesis of ARPD in the future. Although limited to a single case, our results suggest the future possibility of MPM application for the non-invasive diagnosis of skin diseases with connective tissue degeneration, such as ARPD. Funding sources None.
References [1] Rapini RP, Herbert AA, Drucker CR. Acquired perforating dermatosis. Evidence for combined transepidermal elimination of both collagen and elastic fibers. Arch Dermatol 1989;125:1074–8. [2] Tsai TH, Jee SH, Dong CY, Lin SJ. Multiphoton microscopy in dermatological imaging. J Dermatol Sci 2009;56:1–8. [3] Murata T, Honda T, Miyachi Y, Kabashima K. Morphological character of pseudoxanthoma elasticum observed by multiphoton microscopy. J Dermatol Sci 2013;72:199–201. [4] Egawa G, Nakamizo S, Natsuaki Y, Doi H, Miyachi Y, Kabashima K. Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep 2013;3:1932. [5] Egawa G, Natsuaki Y, Miyachi Y, Kabashima K. Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy. J Dermatol Sci 2013;70:143–5. [6] Kabashima K, Egawa G. Intravital multiphoton imaging of cutaneous immune responses. J Invest Dermatol 2014. [7] Koehler MJ, Hahn S, Preller A, Elsner P, Ziemer M, Bauer A, et al. Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network. Exp Dermatol 2008;17:519–23. [8] Tong PL, Qin J, Cooper CL, Lowe PM, Murrell DF, Kossard S, et al. A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy. Br J Dermatol 2013;169:869–79.
Teruasa Murataa,b, Tetsuya Hondaa, Gyohei Egawaa, Yoshiki Miyachia, Kenji Kabashimaa,* a Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; bResearch Fellow of the Japan Society for the Promotion of Science, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3310; fax: +81 75 761 3002 E-mail address: [email protected] (K. Kabashima). Received 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.08.004