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Neuronal hyperexcitability and astrocyte activation in spinal dorsal horn of a dermatitis mouse model with cutaneous hypersensitivity
Neuroscience Letters 720 (2020) 134784
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Research article
Neuronal hyperexcitability and astrocyte activation in spinal dorsal horn of a dermatitis mouse model with cutaneous hypersensitivity
T
Yoshihiro Inamia,b,1,*, Daisuke Utaa,1, Tsugunobu Andoha,** a b
Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Advanced Research Laboratory, Hoyu Co., Ltd., Nagakute, Aichi, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Irritant contact dermatitis Sodium dodecyl sulfate Electrophysiology Spinal dorsal horn Sensory hypersensitivity Astrocytes
Cleaning products such as soaps, shampoos, and detergents are comprised mainly of surfactants, agents known to cause dermatitis and cutaneous hypersensitivity characterized by itching, stinging, and burning of the skin and scalp. However, the mechanisms underlying surfactant-induced cutaneous hypersensitivity remain unclear. In the present study, we investigated the mechanisms of cutaneous hypersensitivity in mice treated with the detergent sodium dodecyl sulfate (SDS). Repeated SDS application to the skin induced inflammation, xeroderma, and elongation of peripheral nerves into the epidermis. The number of neurons immunopositive for c-Fos, a well known marker of neural activity, was substantially higher (+441%) in spinal dorsal horn (SDH) lamina I-II (but not lamina III-VI) of SDS-treated mice compared to vehicle-treated mice. In vivo extracellular recording revealed enhanced spontaneous (+64%) and non-noxious mechanical stimulation-evoked firing (+139%) of SDH lamina I-II neurons in SDS-treated mice, and stimulation-evoked neuronal firing was sustained (+5333%) even after stimulation. The number of GFAP-positive (activated) astrocytes, but not Iba1-positive microglia, was also elevated (+137%) in SDH lamina I-II of SDS-treated mice compared to vehicle-treated mice. Peripheral nerve elongation and hyperexcitability of afferent or SDH neurons, possible associated with the activation of spinal astrocytes, may underlie cutaneous hypersensitivity induced by surfactants.
1. Introduction Cleaning products such as shampoos, soaps, and dishwashing detergents are indispensable items in modern daily life. However, repeated use can cause skin inflammation and cutaneous hypersensitivity reactions in individuals with “sensitive skin,” a subjective cutaneous hyper-reactivity to physical stimuli (ultraviolet radiation, heat, cold, wind), chemicals (cosmetics, soaps, pollution), and even psychological stress. According to three epidemiological studies using self-assessment questionnaires for skin sensitivity conducted in the UK, USA, and France, approximately 60% of women and 40% of men report sensitive skin [1–3]. Cosmetics and soaps are among the candidate triggers according to questionnaire studies [4]. The majority of adverse cutaneous reactions to cleaning products are presumed to be caused by surfactants [5,6]. However, the mechanisms underlying cutaneous hypersensitivity induced by surfactants remain unclear. Sodium dodecyl sulfate (SDS), an anionic surfactant with high
detergency, is widely used in the cleaning products (e.g. 3–50 % w/v: shampoos and skin-cleaning products) [7] and also is used to study irritant contact dermatitis [8,9]. Topical exposure to SDS induces skin inflammation, skin dryness, and cutaneous barrier disruption [8–11]. In addition, we have shown that repeated application of SDS to the rostral dorsal skin of mice induces chronic pruritus and nerve elongation into the epidermis [11,12], suggesting that increased epidermal innervation may be involved in surfactant-induced cutaneous hypersensitivity related to pruritus. However, it is unclear whether this epidermal elongation of peripheral nerves is also involved in the neuronal hyperexcitation associated with persistent skin sensory sensitivity. Therefore, in this study, we measured the spontaneous and stimulus-evoked discharges of spinal dorsal horn (SDH) neurons in a mice model of cutaneous hypersensitivity induced by repeated topical SDS treatment. Furthermore, since it has been shown that spinal astrocytes are involved in chronic itch in mice [13], we also investigated whether these cells are activated in mice treated with SDS.
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Corresponding author at: Advanced Research Laboratory, Hoyu Co., Ltd., 1-12, Roboku, Nagakute, Aichi, 480-1136, Japan. Corresponding author at: Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-1094, Japan. E-mail addresses: [email protected] (Y. Inami), [email protected] (T. Andoh). 1 Authors with equal contributions. ⁎⁎
https://doi.org/10.1016/j.neulet.2020.134784 Received 1 October 2019; Received in revised form 11 January 2020; Accepted 23 January 2020 Available online 24 January 2020 0304-3940/ © 2020 Elsevier B.V. All rights reserved.
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2. Materials and methods
arachnoid membrane to create a window large enough for a tungsten microelectrode, the spinal cord surface was irrigated at 10 ml/min with 95% O2–5% CO2-equilibrated Krebs solution containing (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 11 glucose, and 25 NaHCO3 at 37 ± 1 °C through glass pipettes. Extracellular single-unit recordings of superficial SDH lamina I and II neurons were performed and spikes were selected on amplitude discrimination as described previously [14,15]. The tungsten microelectrode (tip diameter 25 μm, tip impedance 9–12 MΩ) was inserted into the spinal cord of ipsilateral side at an angle of 20–30 degrees (latero-medial) and recordings obtained from neurons 20–100 μm below the surface, which corresponds to lamina I and II. Unit signals were amplified, digitized, and displayed on-line using a special software package (Clampfit version 10.2; Molecular Devices, Union City, CA). We searched for a region on the skin where a touch with a cotton wisp and/or light brush or a noxious pinch with forceps produced a neural response. Neurons were classified as wide dynamic range–type if they responded in a graded manner to innocuous mechanical stimulation and noxious pinch, or nociceptivespecific if they responded to noxious pinch but not to the cotton wisp or brush stimuli. Mechanical stimulation was applied to the right caudal dorsal skin for 5 s using a von Frey filament (vFF; North Coast Medical Inc., Morgan Hill, CA) with a bending force of 0.04 g (non-noxious low force under normal conditions) [16]. After-discharges following the termination of the applied vFF stimulus were also measured for 10 s. For quantification, spontaneous firing rate before stimulation was subtracted from vFF-evoked and after-discharge firing rates.
2.1. Animals Male C57BL/6 mice (Japan SLC, Shizuoka, Japan) were used for experiments at 7 weeks of age. The animals were housed in a room under controlled temperature (22 °C – 23 °C), humidity (45%–65%), and light cycle (lights on from 7:00 AM to 7:00 PM). Food and water were freely available. Procedures used in the animal experiments were approved by the Committee for Animal Experiments at the University of Toyama. 2.2. SDS treatment SDS (Nacalai Tesque, Inc., Kyoto, Japan) was dissolved in distilled water at 10% w/v. The right caudal part of the back was shaved and the remaining hair removed using depilatory cream (Epilat™, Kracie, Tokyo, Japan) at least 3 days prior to the start of the experiment. The 10% SDS solution was applied topically to the entire shaved skin region at 50 μl daily for four consecutive days. 2.3. Measurement of transepidermal water loss (TEWL), stratum corneum (SC) hydration, and dermatitis severity For the evaluation of cutaneous barrier disruption, TEWL (g/m2 per hour) was measured by holding a VapoMeter (model SWL4002; Keystone Scientific K.K., Tokyo, Japan) against the skin for about 20 s. SC hydration (%) was measured using a Moisture Checker MY-808S (Scalar Co., Tokyo, Japan). The severity of dermatitis was scored as follows: 0, no lesion; 1, subtle to mild erythema (< 25% of the hair removal region); 2, moderate erythema (≥ 25% but < 50%); 3, severe erythema (≥ 50%) and hemorrhage.
2.6. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). After testing for normality (Shapiro-Wilk test) and variance equivalency (F-test), group means were compared by Student’s t-test, Welch’s t-test or Mann-Whitney U test, or repeated-measures two-way analysis of variance (RM-ANOVA) followed by post hoc Holm-Šidák tests for pairwise comparisons. A P < 0.05 (two tailed) was considered significant for all tests. All statistical analyses were performed using GraphPad Prism (version 8.0.1; GraphPad Software Inc., San Diego, CA).
2.4. Immunohistochemical staining of skin and spinal cord At 24 h after the final SDS treatment, mice were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. The skin and spinal cord were then removed and embedded in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA) and paraffin, respectively. Then, skin (35-μm thickness) and spinal cord (5-μm thickness) sections were prepared. The following first antibodies were used for the Immunohistochemical staining: rabbit polyclonal anti-protein gene product 9.5 (anti-PGP9.5) antibody (1:1000; Cat# RA95101, Ultraclone, Isle of Wight, UK), rabbit polyclonal anti-c-Fos H-125 (1:100; Cat# sc-7202, Santa Cruz Biotechnology, Santa Cruz, CA), rat polyclonal anti-glial fibrillary acidic protein (GFAP; 1:100; Cat# 130300, Invitrogen Co.), rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:100; Cat# 09–19741, Wako Pure Chemicals, Osaka, Japan), or biotinylated griffonia simplicifolia lectin I isolectin B4 (IB4) (1:100; Cat# B-1205, Vector Laboratories, CA) antibody. The detail methods were shown in Supplemental material.
3. Results 3.1. SDS-induced dermatitis and intra-epidermal nerve elongation Repeated application of 10% SDS to the caudal dorsal skin induced skin inflammation (Fig. 1A and 1B), decreased SC hydration (Fig. 1C), and increased TEWL (Fig. 1D). These skin conditions were significant different after day 2 of SDS treatment compared to vehicle-treated controls (Fig. 1B–D). Epidermal nerve density and the number of epidermal nerve fibers were significantly higher after 4 days in mice receiving repeated application of 10% SDS compared to vehicle controls (Fig. 1E and F).
2.5. In vivo extracellular recordings from mouse SDH 3.2. SDS-induced changes in SDH neural activity as measured by immunostaining
Our previous reports have shown that repeated application of 10% SDS to the rostral back elicited hind-paw scratching and skin inflammation [11]. However, it is difficult to obtain electrophysiological recordings in cervical level of SDH. Therefore, in this study, we treated the caudal dorsal skin with SDS for obtaining electrophysiological results. The methods used for in vivo extracellular recording of superficial SDH neurons were similar to those described previously [14,15]. Briefly, mice were anesthetized with urethane (1.5 g/kg, i.p.). A thoracolumbar laminectomy was performed to expose the spinal column from lumbar 1 (L1) to lumbar 6 (L6) and the animal was then placed in a stereotaxic apparatus. After removing the dura and cutting away the
To examine the distribution of nerves excited by SDS treatment in the SDH, we first performed immunohistochemical staining for c-Fos (a marker of neuronal activity) [17] and IB4 (a marker of the ventral part of lamina I and the dorsal part of lamina II) [18] in sections from L4-5 (Fig. 2A). Although SDS treatment did not affect the number of c-Fosimmunoreactive cells from lamina III to the vicinity of lamina VI, it significantly increased c-Fos-immunoreactive cell number in SDH lamina I-II compared to vehicle treatment (Fig. 2A–C).
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Fig. 1. Changes in stratum corneum (SC) hydration, transepidermal water loss (TEWL), dermatitis score, and immunoreactivity for PGP9.5 in mice during repeated topical application of sodium dodecyl sulfate (SDS). C57BL/6 mice received once daily application of 10% SDS (surfactant) or vehicle (VH, distilled water) to the right caudal dorsal skin for four consecutive days. (A) Dermatitis severity. Pictures of mouse skin after 4 days of treatment with VH or SDS. (B) Daily dermatitis scores. (C) SC hydration. (D) TEWL. (E) Representative images of immunohistochemical staining for PGP9.5 (green) in the skin (scale bar = 100 μm). Inserts depict the regions delineated by the white rectangles at higher magnification (scale bar = 20 μm). The white line and dashed line indicate the skin surface and the border between the epidermis and dermis, respectively: 20× magnification. (F) Quantitative analysis of PGP9.5 immunoreactivity in the epidermis. Values presented as mean ± SEM (n = 10∼11 mice for B–D and 8∼9 mice for F). **p < 0.01 vs. VH (Student’s t-test or Holm-Šidák test).
significantly increased the number of GFAP-immunoreactive astrocytes in SDH lamina I-II of spinal segment L4-5 compared to vehicle-treated mice (Fig. 4B). Furthermore, cell swelling and multiple branching were observed in SDS-treated mice but not vehicle-mice, indicative of astrocytic activation (Fig. 4A). However, SDS treatment did not affect the number or morphology of Iba1-immunoreactive microglia in the same spinal areas (Fig. 4A and C).
3.3. SDS-induced changes in spontaneous and mechanical stimulationevoked firing of superficial SDH neurons Based on immunohistochemical evidence of enhanced neural activity in the SDH of SDS-treated mice (Fig. 2), we recorded spontaneous and stimulus-evoked firing of single neurons in superficial SDH of vehicle- and SDS-treated mice. All neurons recorded in the present study had receptive fields on the right caudal part of the back (the area of vehicle or SDS treatment) as evidenced by enhanced firing rate during touch and/or noxious pinch stimulation. Spontaneous neuronal firing (+64%) in superficial SDH was significantly higher in mice treated with SDS compared to vehicle-treated mice (Fig. 3A and C). Non-painful prick pressure to the right caudal back using a vFF (0.04 g) [16] also evoked higher neuronal firing rates (+139%) in SDS-treated mice compared to vehicle-treated controls (Fig. 3B and D). Furthermore, a conspicuous after-discharge was observed in all SDS-treated mice, and the response was recorded in many neurons=(19/29 from six SDStreated mice) following the termination of the vFF stimulus but not in vehicle-treated controls (Fig. 3B and E). The recording depth of all neurons corresponded to SDH lamina I-II in both vehicle and SDS treatment groups (55.0 ± 4.6 μm and 52.3 ± 3.8 μm, respectively).
4. Discussion In addition to skin inflammation, repetitive SDS treatment to the skin surface induced peripheral nerve elongation into the epidermis and significantly increased both spontaneous and mechanical stimulationevoked neuronal firing in the SDH compared to vehicle-treated control mice. Repetitive cutaneous SDS treatment elicited spontaneous scratching, an itch-related response [11] and was brought skin sensation into the state of “alloknesis” (Supplemental Fig. 1). In this study, we documented activation of SDH astrocytes, a response implicated in cutaneous hypersensitivity and itching. Taken together, these findings suggest that repetitive cutaneous exposure to strong detergents like SDS can trigger skin hypersensitivity through peripheral nerve elongation into the epidermis, sensory and spinal neuron hyperexcitability, and spinal astroglial activation. Repeated SDS treatment to the skin induced elongation of peripheral nerves. Detergents like SDS weaken tight junctions between keratinocytes [21] and induce skin dryness, leading to cutaneous barrier disruption [8–11, present study]. We previously reported that
3.4. SDS-induced activation of spinal cord astrocytes We also conducted immunohistochemical staining for GFAP [19] and Iba1 [20] to assess the contributions of astrocytic and microglial activity to spinal neuron hyperexcitability (Fig. 4A). SDS treatment 3
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Fig. 2. c-Fos and IB4 immunoreactivity in the superficial dorsal horn of lumbar spinal cord segments receiving sensory afferents from treated skin. Spinal cord sections (L4-5 lumbar) collected on day 4 of treatment with VH or SDS were immunostained for c-Fos and IB4. IB4 was used as a regional marker of the ventral part of lamina I and dorsal part of lamina II. (A) Typical images of immunohistochemical staining for c-Fos (green) and IB4 (red) in superficial dorsal horn (ipsilateral side) of L4−5 segments at 20× magnification (scale bar = 100 μm). Dotted line indicates the area of lamina I-II. (B, C) Average number of c-Fos immunoreactive neurons per 100 μm2 area in (B) lamina I-II and (C) lamina III and proximal VI of dorsal horn of the ipsilateral side. Values presented as the mean ± SEM (n = 9 mice per group). Each small circle represents one mouse. **p < 0.01 vs. VH (Mann-Whitney U test).
expression in SDH lamina I-II [33,34]. Therefore, enhanced neuronal firing in SDH lamina I-II may be involved in cutaneous hypersensitivity and itching in SDS-treated mice. Both skin-derived and spinal factors may contribute to SDS-induced spinal neuron hyperactivity. Various factors released from keratinocytes such as histamine may excite elongated peripheral nerve fibers. SDS was shown to increase the epidermal concentration of histamine [11]. In SDH lamina I-II, cutaneous histamine stimulation also increased c-Fos expression [33] and neuronal firing [35,36]. Treatment with SDS also increased the number of astrocytes, but not microglia, in SDH lamina I-II. Furthermore, these astrocytes were activated as evidenced by morphological changes. Activated astrocytes release nitric oxide [37] and prostaglandin E2 [38], both of which induce neuronal firing [39,40]. Therefore, multiple factors released in skin and spinal cord may drive the spontaneous firing of SDH neurons in SDS-treated mice. In addition to spontaneous activity, non-noxious mechanical stimulation-evoked neuronal firing was increased in the superficial SDH of SDS-treated mice. Electrophysiological recordings also revealed a prominent after-discharge in the superficial SDH of SDS-treated mice, but not control mice. These after-discharges may be particularly important for chronic hypersensitivity by conferring sensory input in the absence of actual stimulation. It has been reported that mechanical cutaneous stimulation-induced after-discharges in SDH are due to ectopic afferent activity of primary sensory fibers that persists beyond the stimulus
expression of nerve growth factor (NGF), one of the essential mediators of epidermal nerve fiber sprouting [22,23], was enhanced and that expression of semaphoring 3A (Sema3A), an axon-guidance molecule that inhibits neurite outgrowth of sensory fibers [24,25], was reduced in SDS-treated skin [12]. In addition, we found that histamine concentration and expression of the histamine biosynthetic enzyme histidine decarboxylase (HDC) were increased in epidermis by repetitive SDS treatment [11]. In cultured keratinocytes, histamine induced NGF production [26] and suppressed Sema3A expression [27]. We thus suggest that histamine produced by keratinocytes in response to SDS increases NGF expression and reduces Sema3A expression in the epidermis, which results in nerve fiber elongation into the epidermis. Epidermal nerve elongation has been observed not only in SDS-treated mice but also in atopic dermatitis patients [28] and mice with atopylike dermatitis [29]. In atopic dermatitis patients, cutaneous sensitivity is high [30]. In addition to promoting nerve elongation, NGF also reduces the sensory threshold [31], which would enhance neuronal firing in response to non-noxious stimulation. Taken together, it is suggested that NGF-triggered epidermal elongation contributes to the sensory hypersensitivity of SDS-treated skin. SDS treatment also increased the expression of the neuronal activity marker c-Fos [17] in lamina I-II but not lamina III-VI of the spinal SDH. The C fibers that transmit itch sensation terminate in SDH lamina I-II [32]. In addition, several itch stimuli including allergens increase c-Fos 4
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Fig. 3. Spontaneous firing, mechanical stimulation-evoked firing, and subsequent after-discharges in superficial spinal dorsal horn neurons. After confirming that the recorded neurons received input from the treated skin region by touch or noxious pinch, spontaneous firing was recorded in L4-5 neurons of dorsal horn of the ipsilateral side on day 4 of treatment with VH or SDS. Then, a 0.04-g von Frey filament (vFF) was applied to the skin for 5 s. After-discharges were recorded for 10 s after termination of vFF stimulation. (A, B) Typical representative traces of (A) spontaneous neuronal firing and (B) vFF-evoked neuronal firing and subsequent afterdischarges in L4-5 neurons of VH- and SDS-treated mice. Arrowheads show typical trace of action potential. (C–E) Average rate of (C) spontaneous firing, (D) vFFevoked neuronal firing, and (E) after-discharges in L4-5 neurons. Each small circle represents an action potential recorded in one neuron. The columns and bars represent the mean ± SEM (n = 28 neurons from 5 VH-treated mice and 36 neurons from 6 SDS-treated mice). **p < 0.01 vs. VH (Mann-Whitney U test).
afferents is involved in astrocyte activation within the SDH [13]. These findings suggest that SDS treatment activates spinal astrocytes through activation of TRPV1 channels expressed by primary afferents. It has also been reported that primary afferent-derived transmitters (glutamate, substance P, calcitonin gene-related peptide, and ATP) are involved in the activation of spinal astrocytes [45]. Furthermore, transforming growth factor-β1 (TGF-β1) produced by activated astrocytes may mediate astrocyte migration in spinal cord [46], resulting in altered neural activity.
[41]. This ectopic afferent activity is associated with induction of central nervous system sensitization [42]. The present study did not examine the mechanisms underlying after-discharges. Therefore, the underlying mechanisms will be investigated in future studies. As one of possibility on the mechanisms, however, the properties of SDH cells (neurons and glia cells, especially astrocytes) rather than primary afferent may be involved in the after-discharges. These findings suggest that astrocytes contribute to cutaneous hypersensitivity in SDS-treated mice. Recently, Shiratori-Hayashi et al. [13] reported that lipocalin-2 (LCN2) released from spinal astrocytes contributes to chronic itch and sustained enhancement of itching. LCN2 acts on neurons and several immune cells, including astrocytes, triggering the production of nitric oxide and cytokines [43]. Whether spinal LCN2 is involved in the increased neuronal firing and skin sensitivity of SDS-treated mice warrants further study. Although there are no reports that SDS can directly activate primary afferents, SDS can induce Ca2+ influx via transient receptor potential vanilloid 1 (TRPV1) channels in human neuroblastoma cells [44]. In addition, signaling through TRPV1 channel-expressing primary
5. Conclusions Repeated skin exposure of the surfactant SDS induced peripheral nerve elongation into the epidermis, increased spontaneous and nonnoxious mechanical stimulus-evoked neuronal firing in the SDH, promoted after-discharges of spinal neurons, and activated spinal astrocytes. Collectively, these changes may explain the skin hypersensitivity induced SDS. 5
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Fig. 4. Immunoreactivities of GFAP, Iba1, and IB4 in the superficial dorsal horn of lumbar segments receiving inputs from treated skin. Spinal cord sections (L4-5 lumbar) collected on day 4 of treatment with VH or SDS were immunostained for GFAP, Iba1, and IB4. IB4 (red) was used as regional marker of the ventral part of lamina I and dorsal part of lamina II in dorsal horn. (A) Typical images of immunohistochemical staining for GFAP (green) and Iba1(green) in superficial dorsal horn (ipsilateral side) of L4-5 at 20× magnification (scale bar = 100 μm). Inserts depict higher magnification of the region delineated by the white square (small scale bar = 10 μm). Dotted line indicates the area of lamina I-II. (B, C) Average number of (B) GFAPimmunoreactive cells and (C) Iba1-immunoreactive cells per 100 μm2 area in lamina I-II of L4-5 (dorsal horn of the ipsilateral side). Values presented as mean ± SEM (n = 9 mice). Each small circle represents one mouse. **p < 0.01 vs. VH (Welch's t-test).
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Author contributions
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Y.I. and D.U. designed and performed most of the experiments and analyzed the data. Y.I and T.A. conceived the study and supervised the overall project. Y.I., D.U., and T.A. wrote the manuscript. All of the authors have read and approved the final version of the manuscript. Declaration of Competing Interest Dr. Inami, Yosuke Mano, and Miki Fukushima are employed by Hoyu Co., Ltd. who financially supported the research. Acknowlegments Funding for this study was provided by a grant from Hoyu Co., Ltd (Nagakute, Aichi, Japan). We would like to thank Yosuke Mano and Miki Fukushima of Hoyu Co., Ltd. for assistance with histology. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2020.134784. References [1] C.M. Willis, S. Shaw, O. De Lacharrière, M. Baverel, L. Reiche, R. Jourdain, P. Bastien, J.D. Wilkinson, Sensitive skin: an epidemiological study, Br. J. Dermatol. 145 (2) (2001) 258–263. [2] R. Jourdain, O. de Lacharrière, P. Bastien, H.I. Maibach, Ethnic variations in selfperceived sensitive skin: epidemiological survey, Contact Derm. 46 (3) (2002) 162–169. [3] C. Guinot, D. Malvy, E. Mauger, K. Ezzedine, J. Latreille, L. Ambroisine, M. Tenenhaus, P. Préziosi, F. Morizot, P. Galan, S. Hercberg, E. Tschachler, Selfreported skin sensitivity in a general adult population in France: data of the SU.VI.MAX cohort, J. Eur. Acad. Dermatol. Venereol. 20 (4) (2006) 380–390. [4] C. Saint-Martory, A.M. Roguedas-Contios, V. Sibaud, A. Degouy, A.M. Schmitt, L. Misery, Sensitive skin is not limited to the face, Br. J. Dermatol. 158 (1) (2008) 130–133. [5] A.C. Groot, J.P. Nater, R. Van der Lende, B. Bijcken, Adverse effects of cosmetics and toiletries: a retrospective study in the general population, Int. J. Cosmet. Sci. 9 (6) (1987) 255–259, https://doi.org/10.1111/j.1467-2494.1987.tb00481.x. [6] B. Berne, A. Bostrom, A.F. Grahnen, M. Tammela, Adverse effects of cosmetics and toiletries reported to the Swedish Medical Products Agency 1989-1994, Contact Derm. 34 (5) (1996) 359–362. [7] Cosmetic Ingredient Review Expert Panel, Annual review of cosmetic ingredient safety assessments–2002/2003, Int. J. Toxicol. 24 (Suppl. 1) (2005) 1–102, https:// doi.org/10.1080/10915810590918625. [8] T. Agner, J. Serup, Sodium lauryl sulphate for irritant patch testing–a dose–response study using bioengineering methods for determination of skin irritation, J. Invest. Dermatol. 95 (5) (1990) 543–547. [9] I. Effendy, H.I. Maibach, Surfactants and experimental irritant contact dermatitis, Contact Derm. 33 (4) (1995) 217–225. [10] M. Le, J. Schalkwijk, G. Siegenthaler, P.C. van de Kerkhof, J.H. Veerkamp, P.G. van der Valk, Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate, Arch. Dermatol. Res. 288 (11) (1996) 684–690. [11] Y. Inami, A. Sasaki, T. Andoh, Y. Kuraishi, Surfactant-induced chronic pruritus: role of L-histidine decarboxylase expression and histamine production in epidermis, Acta Derm. Venereol. 94 (6) (2014) 645–650, https://doi.org/10.2340/000155551834. [12] Y. Inami, A. Sato, H. Ohtsu, Y. Mano, A. Sasaki, Y. Kuraishi, T. Andoh, Deficiency of histidine decarboxylase attenuates peripheral nerve fibre elongation into the epidermis in surfactant-treated mouse skin, Exp. Dermatol. 26 (5) (2017) 441–443, https://doi.org/10.1111/exd.13223. [13] M. Shiratori-Hayashi, K. Koga, H. Tozaki-Saitoh, Y. Kohro, H. Toyonaga, C. Yamaguchi, A. Hasegawa, T. Nakahara, J. Hachisuka, S. Akira, H. Okano, M. Furue, K. Inoue, M. Tsuda, STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch, Nat. Med. 21 (8) (2015) 927–931, https://doi. org/10.1038/nm.3912. [14] T. Andoh, D. Uta, M. Kato, K. Toume, K. Komatsu, Y. Kuraishi, Prophylactic administration of aucubin inhibits paclitaxel-induced mechanical allodynia via the inhibition of endoplasmic reticulum stress in peripheral schwann cells, Biol. Pharm. Bull. 40 (4) (2017) 473–478, https://doi.org/10.1248/bpb.b16-00899. [15] T. Akiyama, A.W. Merrill, M.I. Carstens, E. Carstens, Activation of superficial dorsal horn neurons in the mouse by a PAR-2 agonist and 5-HT: potential role in itch, J. Neurosci. 29 (20) (2009) 6691–6699, https://doi.org/10.1523/JNEUROSCI.610308.2009. [16] T. Akiyama, M.I. Carstens, A. Ikoma, F. Cevikbas, M. Steinhoff, E. Carstens, Mouse model of touch-evoked itch (alloknesis), J. Invest. Dermatol. 132 (7) (2012) 1886–1891, https://doi.org/10.1038/jid.2012.52.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Neuronal hyperexcitability and astrocyte activation in spinal dorsal horn of a dermatitis mouse model with cutaneous hypersensitivity
T
Yoshihiro Inamia,b,1,*, Daisuke Utaa,1, Tsugunobu Andoha,** a b
Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Advanced Research Laboratory, Hoyu Co., Ltd., Nagakute, Aichi, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Irritant contact dermatitis Sodium dodecyl sulfate Electrophysiology Spinal dorsal horn Sensory hypersensitivity Astrocytes
Cleaning products such as soaps, shampoos, and detergents are comprised mainly of surfactants, agents known to cause dermatitis and cutaneous hypersensitivity characterized by itching, stinging, and burning of the skin and scalp. However, the mechanisms underlying surfactant-induced cutaneous hypersensitivity remain unclear. In the present study, we investigated the mechanisms of cutaneous hypersensitivity in mice treated with the detergent sodium dodecyl sulfate (SDS). Repeated SDS application to the skin induced inflammation, xeroderma, and elongation of peripheral nerves into the epidermis. The number of neurons immunopositive for c-Fos, a well known marker of neural activity, was substantially higher (+441%) in spinal dorsal horn (SDH) lamina I-II (but not lamina III-VI) of SDS-treated mice compared to vehicle-treated mice. In vivo extracellular recording revealed enhanced spontaneous (+64%) and non-noxious mechanical stimulation-evoked firing (+139%) of SDH lamina I-II neurons in SDS-treated mice, and stimulation-evoked neuronal firing was sustained (+5333%) even after stimulation. The number of GFAP-positive (activated) astrocytes, but not Iba1-positive microglia, was also elevated (+137%) in SDH lamina I-II of SDS-treated mice compared to vehicle-treated mice. Peripheral nerve elongation and hyperexcitability of afferent or SDH neurons, possible associated with the activation of spinal astrocytes, may underlie cutaneous hypersensitivity induced by surfactants.
1. Introduction Cleaning products such as shampoos, soaps, and dishwashing detergents are indispensable items in modern daily life. However, repeated use can cause skin inflammation and cutaneous hypersensitivity reactions in individuals with “sensitive skin,” a subjective cutaneous hyper-reactivity to physical stimuli (ultraviolet radiation, heat, cold, wind), chemicals (cosmetics, soaps, pollution), and even psychological stress. According to three epidemiological studies using self-assessment questionnaires for skin sensitivity conducted in the UK, USA, and France, approximately 60% of women and 40% of men report sensitive skin [1–3]. Cosmetics and soaps are among the candidate triggers according to questionnaire studies [4]. The majority of adverse cutaneous reactions to cleaning products are presumed to be caused by surfactants [5,6]. However, the mechanisms underlying cutaneous hypersensitivity induced by surfactants remain unclear. Sodium dodecyl sulfate (SDS), an anionic surfactant with high
detergency, is widely used in the cleaning products (e.g. 3–50 % w/v: shampoos and skin-cleaning products) [7] and also is used to study irritant contact dermatitis [8,9]. Topical exposure to SDS induces skin inflammation, skin dryness, and cutaneous barrier disruption [8–11]. In addition, we have shown that repeated application of SDS to the rostral dorsal skin of mice induces chronic pruritus and nerve elongation into the epidermis [11,12], suggesting that increased epidermal innervation may be involved in surfactant-induced cutaneous hypersensitivity related to pruritus. However, it is unclear whether this epidermal elongation of peripheral nerves is also involved in the neuronal hyperexcitation associated with persistent skin sensory sensitivity. Therefore, in this study, we measured the spontaneous and stimulus-evoked discharges of spinal dorsal horn (SDH) neurons in a mice model of cutaneous hypersensitivity induced by repeated topical SDS treatment. Furthermore, since it has been shown that spinal astrocytes are involved in chronic itch in mice [13], we also investigated whether these cells are activated in mice treated with SDS.
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Corresponding author at: Advanced Research Laboratory, Hoyu Co., Ltd., 1-12, Roboku, Nagakute, Aichi, 480-1136, Japan. Corresponding author at: Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-1094, Japan. E-mail addresses: [email protected] (Y. Inami), [email protected] (T. Andoh). 1 Authors with equal contributions. ⁎⁎
https://doi.org/10.1016/j.neulet.2020.134784 Received 1 October 2019; Received in revised form 11 January 2020; Accepted 23 January 2020 Available online 24 January 2020 0304-3940/ © 2020 Elsevier B.V. All rights reserved.
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2. Materials and methods
arachnoid membrane to create a window large enough for a tungsten microelectrode, the spinal cord surface was irrigated at 10 ml/min with 95% O2–5% CO2-equilibrated Krebs solution containing (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 11 glucose, and 25 NaHCO3 at 37 ± 1 °C through glass pipettes. Extracellular single-unit recordings of superficial SDH lamina I and II neurons were performed and spikes were selected on amplitude discrimination as described previously [14,15]. The tungsten microelectrode (tip diameter 25 μm, tip impedance 9–12 MΩ) was inserted into the spinal cord of ipsilateral side at an angle of 20–30 degrees (latero-medial) and recordings obtained from neurons 20–100 μm below the surface, which corresponds to lamina I and II. Unit signals were amplified, digitized, and displayed on-line using a special software package (Clampfit version 10.2; Molecular Devices, Union City, CA). We searched for a region on the skin where a touch with a cotton wisp and/or light brush or a noxious pinch with forceps produced a neural response. Neurons were classified as wide dynamic range–type if they responded in a graded manner to innocuous mechanical stimulation and noxious pinch, or nociceptivespecific if they responded to noxious pinch but not to the cotton wisp or brush stimuli. Mechanical stimulation was applied to the right caudal dorsal skin for 5 s using a von Frey filament (vFF; North Coast Medical Inc., Morgan Hill, CA) with a bending force of 0.04 g (non-noxious low force under normal conditions) [16]. After-discharges following the termination of the applied vFF stimulus were also measured for 10 s. For quantification, spontaneous firing rate before stimulation was subtracted from vFF-evoked and after-discharge firing rates.
2.1. Animals Male C57BL/6 mice (Japan SLC, Shizuoka, Japan) were used for experiments at 7 weeks of age. The animals were housed in a room under controlled temperature (22 °C – 23 °C), humidity (45%–65%), and light cycle (lights on from 7:00 AM to 7:00 PM). Food and water were freely available. Procedures used in the animal experiments were approved by the Committee for Animal Experiments at the University of Toyama. 2.2. SDS treatment SDS (Nacalai Tesque, Inc., Kyoto, Japan) was dissolved in distilled water at 10% w/v. The right caudal part of the back was shaved and the remaining hair removed using depilatory cream (Epilat™, Kracie, Tokyo, Japan) at least 3 days prior to the start of the experiment. The 10% SDS solution was applied topically to the entire shaved skin region at 50 μl daily for four consecutive days. 2.3. Measurement of transepidermal water loss (TEWL), stratum corneum (SC) hydration, and dermatitis severity For the evaluation of cutaneous barrier disruption, TEWL (g/m2 per hour) was measured by holding a VapoMeter (model SWL4002; Keystone Scientific K.K., Tokyo, Japan) against the skin for about 20 s. SC hydration (%) was measured using a Moisture Checker MY-808S (Scalar Co., Tokyo, Japan). The severity of dermatitis was scored as follows: 0, no lesion; 1, subtle to mild erythema (< 25% of the hair removal region); 2, moderate erythema (≥ 25% but < 50%); 3, severe erythema (≥ 50%) and hemorrhage.
2.6. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). After testing for normality (Shapiro-Wilk test) and variance equivalency (F-test), group means were compared by Student’s t-test, Welch’s t-test or Mann-Whitney U test, or repeated-measures two-way analysis of variance (RM-ANOVA) followed by post hoc Holm-Šidák tests for pairwise comparisons. A P < 0.05 (two tailed) was considered significant for all tests. All statistical analyses were performed using GraphPad Prism (version 8.0.1; GraphPad Software Inc., San Diego, CA).
2.4. Immunohistochemical staining of skin and spinal cord At 24 h after the final SDS treatment, mice were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. The skin and spinal cord were then removed and embedded in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA) and paraffin, respectively. Then, skin (35-μm thickness) and spinal cord (5-μm thickness) sections were prepared. The following first antibodies were used for the Immunohistochemical staining: rabbit polyclonal anti-protein gene product 9.5 (anti-PGP9.5) antibody (1:1000; Cat# RA95101, Ultraclone, Isle of Wight, UK), rabbit polyclonal anti-c-Fos H-125 (1:100; Cat# sc-7202, Santa Cruz Biotechnology, Santa Cruz, CA), rat polyclonal anti-glial fibrillary acidic protein (GFAP; 1:100; Cat# 130300, Invitrogen Co.), rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:100; Cat# 09–19741, Wako Pure Chemicals, Osaka, Japan), or biotinylated griffonia simplicifolia lectin I isolectin B4 (IB4) (1:100; Cat# B-1205, Vector Laboratories, CA) antibody. The detail methods were shown in Supplemental material.
3. Results 3.1. SDS-induced dermatitis and intra-epidermal nerve elongation Repeated application of 10% SDS to the caudal dorsal skin induced skin inflammation (Fig. 1A and 1B), decreased SC hydration (Fig. 1C), and increased TEWL (Fig. 1D). These skin conditions were significant different after day 2 of SDS treatment compared to vehicle-treated controls (Fig. 1B–D). Epidermal nerve density and the number of epidermal nerve fibers were significantly higher after 4 days in mice receiving repeated application of 10% SDS compared to vehicle controls (Fig. 1E and F).
2.5. In vivo extracellular recordings from mouse SDH 3.2. SDS-induced changes in SDH neural activity as measured by immunostaining
Our previous reports have shown that repeated application of 10% SDS to the rostral back elicited hind-paw scratching and skin inflammation [11]. However, it is difficult to obtain electrophysiological recordings in cervical level of SDH. Therefore, in this study, we treated the caudal dorsal skin with SDS for obtaining electrophysiological results. The methods used for in vivo extracellular recording of superficial SDH neurons were similar to those described previously [14,15]. Briefly, mice were anesthetized with urethane (1.5 g/kg, i.p.). A thoracolumbar laminectomy was performed to expose the spinal column from lumbar 1 (L1) to lumbar 6 (L6) and the animal was then placed in a stereotaxic apparatus. After removing the dura and cutting away the
To examine the distribution of nerves excited by SDS treatment in the SDH, we first performed immunohistochemical staining for c-Fos (a marker of neuronal activity) [17] and IB4 (a marker of the ventral part of lamina I and the dorsal part of lamina II) [18] in sections from L4-5 (Fig. 2A). Although SDS treatment did not affect the number of c-Fosimmunoreactive cells from lamina III to the vicinity of lamina VI, it significantly increased c-Fos-immunoreactive cell number in SDH lamina I-II compared to vehicle treatment (Fig. 2A–C).
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Fig. 1. Changes in stratum corneum (SC) hydration, transepidermal water loss (TEWL), dermatitis score, and immunoreactivity for PGP9.5 in mice during repeated topical application of sodium dodecyl sulfate (SDS). C57BL/6 mice received once daily application of 10% SDS (surfactant) or vehicle (VH, distilled water) to the right caudal dorsal skin for four consecutive days. (A) Dermatitis severity. Pictures of mouse skin after 4 days of treatment with VH or SDS. (B) Daily dermatitis scores. (C) SC hydration. (D) TEWL. (E) Representative images of immunohistochemical staining for PGP9.5 (green) in the skin (scale bar = 100 μm). Inserts depict the regions delineated by the white rectangles at higher magnification (scale bar = 20 μm). The white line and dashed line indicate the skin surface and the border between the epidermis and dermis, respectively: 20× magnification. (F) Quantitative analysis of PGP9.5 immunoreactivity in the epidermis. Values presented as mean ± SEM (n = 10∼11 mice for B–D and 8∼9 mice for F). **p < 0.01 vs. VH (Student’s t-test or Holm-Šidák test).
significantly increased the number of GFAP-immunoreactive astrocytes in SDH lamina I-II of spinal segment L4-5 compared to vehicle-treated mice (Fig. 4B). Furthermore, cell swelling and multiple branching were observed in SDS-treated mice but not vehicle-mice, indicative of astrocytic activation (Fig. 4A). However, SDS treatment did not affect the number or morphology of Iba1-immunoreactive microglia in the same spinal areas (Fig. 4A and C).
3.3. SDS-induced changes in spontaneous and mechanical stimulationevoked firing of superficial SDH neurons Based on immunohistochemical evidence of enhanced neural activity in the SDH of SDS-treated mice (Fig. 2), we recorded spontaneous and stimulus-evoked firing of single neurons in superficial SDH of vehicle- and SDS-treated mice. All neurons recorded in the present study had receptive fields on the right caudal part of the back (the area of vehicle or SDS treatment) as evidenced by enhanced firing rate during touch and/or noxious pinch stimulation. Spontaneous neuronal firing (+64%) in superficial SDH was significantly higher in mice treated with SDS compared to vehicle-treated mice (Fig. 3A and C). Non-painful prick pressure to the right caudal back using a vFF (0.04 g) [16] also evoked higher neuronal firing rates (+139%) in SDS-treated mice compared to vehicle-treated controls (Fig. 3B and D). Furthermore, a conspicuous after-discharge was observed in all SDS-treated mice, and the response was recorded in many neurons=(19/29 from six SDStreated mice) following the termination of the vFF stimulus but not in vehicle-treated controls (Fig. 3B and E). The recording depth of all neurons corresponded to SDH lamina I-II in both vehicle and SDS treatment groups (55.0 ± 4.6 μm and 52.3 ± 3.8 μm, respectively).
4. Discussion In addition to skin inflammation, repetitive SDS treatment to the skin surface induced peripheral nerve elongation into the epidermis and significantly increased both spontaneous and mechanical stimulationevoked neuronal firing in the SDH compared to vehicle-treated control mice. Repetitive cutaneous SDS treatment elicited spontaneous scratching, an itch-related response [11] and was brought skin sensation into the state of “alloknesis” (Supplemental Fig. 1). In this study, we documented activation of SDH astrocytes, a response implicated in cutaneous hypersensitivity and itching. Taken together, these findings suggest that repetitive cutaneous exposure to strong detergents like SDS can trigger skin hypersensitivity through peripheral nerve elongation into the epidermis, sensory and spinal neuron hyperexcitability, and spinal astroglial activation. Repeated SDS treatment to the skin induced elongation of peripheral nerves. Detergents like SDS weaken tight junctions between keratinocytes [21] and induce skin dryness, leading to cutaneous barrier disruption [8–11, present study]. We previously reported that
3.4. SDS-induced activation of spinal cord astrocytes We also conducted immunohistochemical staining for GFAP [19] and Iba1 [20] to assess the contributions of astrocytic and microglial activity to spinal neuron hyperexcitability (Fig. 4A). SDS treatment 3
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Fig. 2. c-Fos and IB4 immunoreactivity in the superficial dorsal horn of lumbar spinal cord segments receiving sensory afferents from treated skin. Spinal cord sections (L4-5 lumbar) collected on day 4 of treatment with VH or SDS were immunostained for c-Fos and IB4. IB4 was used as a regional marker of the ventral part of lamina I and dorsal part of lamina II. (A) Typical images of immunohistochemical staining for c-Fos (green) and IB4 (red) in superficial dorsal horn (ipsilateral side) of L4−5 segments at 20× magnification (scale bar = 100 μm). Dotted line indicates the area of lamina I-II. (B, C) Average number of c-Fos immunoreactive neurons per 100 μm2 area in (B) lamina I-II and (C) lamina III and proximal VI of dorsal horn of the ipsilateral side. Values presented as the mean ± SEM (n = 9 mice per group). Each small circle represents one mouse. **p < 0.01 vs. VH (Mann-Whitney U test).
expression in SDH lamina I-II [33,34]. Therefore, enhanced neuronal firing in SDH lamina I-II may be involved in cutaneous hypersensitivity and itching in SDS-treated mice. Both skin-derived and spinal factors may contribute to SDS-induced spinal neuron hyperactivity. Various factors released from keratinocytes such as histamine may excite elongated peripheral nerve fibers. SDS was shown to increase the epidermal concentration of histamine [11]. In SDH lamina I-II, cutaneous histamine stimulation also increased c-Fos expression [33] and neuronal firing [35,36]. Treatment with SDS also increased the number of astrocytes, but not microglia, in SDH lamina I-II. Furthermore, these astrocytes were activated as evidenced by morphological changes. Activated astrocytes release nitric oxide [37] and prostaglandin E2 [38], both of which induce neuronal firing [39,40]. Therefore, multiple factors released in skin and spinal cord may drive the spontaneous firing of SDH neurons in SDS-treated mice. In addition to spontaneous activity, non-noxious mechanical stimulation-evoked neuronal firing was increased in the superficial SDH of SDS-treated mice. Electrophysiological recordings also revealed a prominent after-discharge in the superficial SDH of SDS-treated mice, but not control mice. These after-discharges may be particularly important for chronic hypersensitivity by conferring sensory input in the absence of actual stimulation. It has been reported that mechanical cutaneous stimulation-induced after-discharges in SDH are due to ectopic afferent activity of primary sensory fibers that persists beyond the stimulus
expression of nerve growth factor (NGF), one of the essential mediators of epidermal nerve fiber sprouting [22,23], was enhanced and that expression of semaphoring 3A (Sema3A), an axon-guidance molecule that inhibits neurite outgrowth of sensory fibers [24,25], was reduced in SDS-treated skin [12]. In addition, we found that histamine concentration and expression of the histamine biosynthetic enzyme histidine decarboxylase (HDC) were increased in epidermis by repetitive SDS treatment [11]. In cultured keratinocytes, histamine induced NGF production [26] and suppressed Sema3A expression [27]. We thus suggest that histamine produced by keratinocytes in response to SDS increases NGF expression and reduces Sema3A expression in the epidermis, which results in nerve fiber elongation into the epidermis. Epidermal nerve elongation has been observed not only in SDS-treated mice but also in atopic dermatitis patients [28] and mice with atopylike dermatitis [29]. In atopic dermatitis patients, cutaneous sensitivity is high [30]. In addition to promoting nerve elongation, NGF also reduces the sensory threshold [31], which would enhance neuronal firing in response to non-noxious stimulation. Taken together, it is suggested that NGF-triggered epidermal elongation contributes to the sensory hypersensitivity of SDS-treated skin. SDS treatment also increased the expression of the neuronal activity marker c-Fos [17] in lamina I-II but not lamina III-VI of the spinal SDH. The C fibers that transmit itch sensation terminate in SDH lamina I-II [32]. In addition, several itch stimuli including allergens increase c-Fos 4
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Fig. 3. Spontaneous firing, mechanical stimulation-evoked firing, and subsequent after-discharges in superficial spinal dorsal horn neurons. After confirming that the recorded neurons received input from the treated skin region by touch or noxious pinch, spontaneous firing was recorded in L4-5 neurons of dorsal horn of the ipsilateral side on day 4 of treatment with VH or SDS. Then, a 0.04-g von Frey filament (vFF) was applied to the skin for 5 s. After-discharges were recorded for 10 s after termination of vFF stimulation. (A, B) Typical representative traces of (A) spontaneous neuronal firing and (B) vFF-evoked neuronal firing and subsequent afterdischarges in L4-5 neurons of VH- and SDS-treated mice. Arrowheads show typical trace of action potential. (C–E) Average rate of (C) spontaneous firing, (D) vFFevoked neuronal firing, and (E) after-discharges in L4-5 neurons. Each small circle represents an action potential recorded in one neuron. The columns and bars represent the mean ± SEM (n = 28 neurons from 5 VH-treated mice and 36 neurons from 6 SDS-treated mice). **p < 0.01 vs. VH (Mann-Whitney U test).
afferents is involved in astrocyte activation within the SDH [13]. These findings suggest that SDS treatment activates spinal astrocytes through activation of TRPV1 channels expressed by primary afferents. It has also been reported that primary afferent-derived transmitters (glutamate, substance P, calcitonin gene-related peptide, and ATP) are involved in the activation of spinal astrocytes [45]. Furthermore, transforming growth factor-β1 (TGF-β1) produced by activated astrocytes may mediate astrocyte migration in spinal cord [46], resulting in altered neural activity.
[41]. This ectopic afferent activity is associated with induction of central nervous system sensitization [42]. The present study did not examine the mechanisms underlying after-discharges. Therefore, the underlying mechanisms will be investigated in future studies. As one of possibility on the mechanisms, however, the properties of SDH cells (neurons and glia cells, especially astrocytes) rather than primary afferent may be involved in the after-discharges. These findings suggest that astrocytes contribute to cutaneous hypersensitivity in SDS-treated mice. Recently, Shiratori-Hayashi et al. [13] reported that lipocalin-2 (LCN2) released from spinal astrocytes contributes to chronic itch and sustained enhancement of itching. LCN2 acts on neurons and several immune cells, including astrocytes, triggering the production of nitric oxide and cytokines [43]. Whether spinal LCN2 is involved in the increased neuronal firing and skin sensitivity of SDS-treated mice warrants further study. Although there are no reports that SDS can directly activate primary afferents, SDS can induce Ca2+ influx via transient receptor potential vanilloid 1 (TRPV1) channels in human neuroblastoma cells [44]. In addition, signaling through TRPV1 channel-expressing primary
5. Conclusions Repeated skin exposure of the surfactant SDS induced peripheral nerve elongation into the epidermis, increased spontaneous and nonnoxious mechanical stimulus-evoked neuronal firing in the SDH, promoted after-discharges of spinal neurons, and activated spinal astrocytes. Collectively, these changes may explain the skin hypersensitivity induced SDS. 5
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Fig. 4. Immunoreactivities of GFAP, Iba1, and IB4 in the superficial dorsal horn of lumbar segments receiving inputs from treated skin. Spinal cord sections (L4-5 lumbar) collected on day 4 of treatment with VH or SDS were immunostained for GFAP, Iba1, and IB4. IB4 (red) was used as regional marker of the ventral part of lamina I and dorsal part of lamina II in dorsal horn. (A) Typical images of immunohistochemical staining for GFAP (green) and Iba1(green) in superficial dorsal horn (ipsilateral side) of L4-5 at 20× magnification (scale bar = 100 μm). Inserts depict higher magnification of the region delineated by the white square (small scale bar = 10 μm). Dotted line indicates the area of lamina I-II. (B, C) Average number of (B) GFAPimmunoreactive cells and (C) Iba1-immunoreactive cells per 100 μm2 area in lamina I-II of L4-5 (dorsal horn of the ipsilateral side). Values presented as mean ± SEM (n = 9 mice). Each small circle represents one mouse. **p < 0.01 vs. VH (Welch's t-test).
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Author contributions
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Y.I. and D.U. designed and performed most of the experiments and analyzed the data. Y.I and T.A. conceived the study and supervised the overall project. Y.I., D.U., and T.A. wrote the manuscript. All of the authors have read and approved the final version of the manuscript. Declaration of Competing Interest Dr. Inami, Yosuke Mano, and Miki Fukushima are employed by Hoyu Co., Ltd. who financially supported the research. Acknowlegments Funding for this study was provided by a grant from Hoyu Co., Ltd (Nagakute, Aichi, Japan). We would like to thank Yosuke Mano and Miki Fukushima of Hoyu Co., Ltd. for assistance with histology. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2020.134784. References [1] C.M. Willis, S. Shaw, O. De Lacharrière, M. Baverel, L. Reiche, R. Jourdain, P. Bastien, J.D. Wilkinson, Sensitive skin: an epidemiological study, Br. J. Dermatol. 145 (2) (2001) 258–263. [2] R. Jourdain, O. de Lacharrière, P. Bastien, H.I. Maibach, Ethnic variations in selfperceived sensitive skin: epidemiological survey, Contact Derm. 46 (3) (2002) 162–169. [3] C. Guinot, D. Malvy, E. Mauger, K. Ezzedine, J. Latreille, L. Ambroisine, M. Tenenhaus, P. Préziosi, F. Morizot, P. Galan, S. Hercberg, E. Tschachler, Selfreported skin sensitivity in a general adult population in France: data of the SU.VI.MAX cohort, J. Eur. Acad. Dermatol. Venereol. 20 (4) (2006) 380–390. [4] C. Saint-Martory, A.M. Roguedas-Contios, V. Sibaud, A. Degouy, A.M. Schmitt, L. Misery, Sensitive skin is not limited to the face, Br. J. Dermatol. 158 (1) (2008) 130–133. [5] A.C. Groot, J.P. Nater, R. Van der Lende, B. Bijcken, Adverse effects of cosmetics and toiletries: a retrospective study in the general population, Int. J. Cosmet. Sci. 9 (6) (1987) 255–259, https://doi.org/10.1111/j.1467-2494.1987.tb00481.x. [6] B. Berne, A. Bostrom, A.F. Grahnen, M. Tammela, Adverse effects of cosmetics and toiletries reported to the Swedish Medical Products Agency 1989-1994, Contact Derm. 34 (5) (1996) 359–362. [7] Cosmetic Ingredient Review Expert Panel, Annual review of cosmetic ingredient safety assessments–2002/2003, Int. J. Toxicol. 24 (Suppl. 1) (2005) 1–102, https:// doi.org/10.1080/10915810590918625. [8] T. Agner, J. Serup, Sodium lauryl sulphate for irritant patch testing–a dose–response study using bioengineering methods for determination of skin irritation, J. Invest. Dermatol. 95 (5) (1990) 543–547. [9] I. Effendy, H.I. Maibach, Surfactants and experimental irritant contact dermatitis, Contact Derm. 33 (4) (1995) 217–225. [10] M. Le, J. Schalkwijk, G. Siegenthaler, P.C. van de Kerkhof, J.H. Veerkamp, P.G. van der Valk, Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate, Arch. Dermatol. Res. 288 (11) (1996) 684–690. [11] Y. Inami, A. Sasaki, T. Andoh, Y. Kuraishi, Surfactant-induced chronic pruritus: role of L-histidine decarboxylase expression and histamine production in epidermis, Acta Derm. Venereol. 94 (6) (2014) 645–650, https://doi.org/10.2340/000155551834. [12] Y. Inami, A. Sato, H. Ohtsu, Y. Mano, A. Sasaki, Y. Kuraishi, T. Andoh, Deficiency of histidine decarboxylase attenuates peripheral nerve fibre elongation into the epidermis in surfactant-treated mouse skin, Exp. Dermatol. 26 (5) (2017) 441–443, https://doi.org/10.1111/exd.13223. [13] M. Shiratori-Hayashi, K. Koga, H. Tozaki-Saitoh, Y. Kohro, H. Toyonaga, C. Yamaguchi, A. Hasegawa, T. Nakahara, J. Hachisuka, S. Akira, H. Okano, M. Furue, K. Inoue, M. Tsuda, STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch, Nat. Med. 21 (8) (2015) 927–931, https://doi. org/10.1038/nm.3912. [14] T. Andoh, D. Uta, M. Kato, K. Toume, K. Komatsu, Y. Kuraishi, Prophylactic administration of aucubin inhibits paclitaxel-induced mechanical allodynia via the inhibition of endoplasmic reticulum stress in peripheral schwann cells, Biol. Pharm. Bull. 40 (4) (2017) 473–478, https://doi.org/10.1248/bpb.b16-00899. [15] T. Akiyama, A.W. Merrill, M.I. Carstens, E. Carstens, Activation of superficial dorsal horn neurons in the mouse by a PAR-2 agonist and 5-HT: potential role in itch, J. Neurosci. 29 (20) (2009) 6691–6699, https://doi.org/10.1523/JNEUROSCI.610308.2009. [16] T. Akiyama, M.I. Carstens, A. Ikoma, F. Cevikbas, M. Steinhoff, E. Carstens, Mouse model of touch-evoked itch (alloknesis), J. Invest. Dermatol. 132 (7) (2012) 1886–1891, https://doi.org/10.1038/jid.2012.52.
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