INTRAEPIDERMAL NERVE FIBER DENSITY IN VULVAR LICHEN
SCLEROSUS AND NORMAL VULVAR TISSUES

Lichen sclerosus (LS) is a chronic and inflammatory disease of genital and extragenital skin in women and less frequently in men with a potential for functional deterioration, atrophy, destructive scarring, and possibly malignant evolution (1). Its pathogenesis is not yet fully elucidated and is probably multifactorial. Autoimmune mechanisms have been implicated due to strong relation of the disease with autoantibodies targeted against the extracellular matrix protein 1 (ECM1) (2) and to a lesser extent against components of the basement membrane zone (BMZ) (3). Association with other autoimmune diseases such as thyroiditis, alopecia areata, pernicious anemia, and vitiligo has been also observed (4). Recent studies on pathogenesis of lichenoid dermatoses with postulated autoimmune background (5, 6) suggest that oxidative stress may also be involved in etiology of LS (7). Moreover, a genetic susceptibility as well as trauma and chronic irritation could be shown to be provocative (1). In addition, as the disease tends to appear during periods of life when the estrogen influence is lower, hormonal influences should not be neglected in the etiology (4). Infectious pathogens such as Epstein-Barr-Virus and Borrellia burgdorferi could not be consistently linked to LS (1).

LS in genital area is characterized by an array of sensory sensations such as pain and soreness of the vulvar and perianal areas, dysuria, dyspareunia, and pain on defecation, but intractable itch is often mentioned in the first place (4). Pathophysiology of the most chronic itch conditions is not fully explained. A mechanism-based definition of itch distinguishes itch sensation evoked in healthy nervous system by peripheral (pruriceptive) and central (neurogenic) mechanisms and itch that is caused by diseased neurons (neuropathic itch) (8). Two peripheral itch-sensitive pathways are described as follows: a histamine-stimulated pathway that uses mechanically insensitive C fibers and a so-called cowhage-stimulated pathway, primarily involving mechanically sensitive C fibers (9, 10). In skin disorders, histamine-dependent mechanism and inflammatory mediators are believed to play crucial role in evoking itch sensation (8). But, there is growing evidence, for example, in atopic dermatitis (11), showing that nonhistaminergic mechanisms are also involved in eliciting itch sensation in pruritic conditions. Modern concept of neurogenic inflammation explains interaction between cutaneous nerve fibers, skin, and immune system mediated by neuropeptides, neurotransmitters, neurotrophins, and neurohormones that influence a variety of physiological and pathophysiological phenomena including itch sensation (12).

Numerous mediators of skin cells can activate and sensitize pruritic nerve fibers and even modulate their growth (13). Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are neuropeptides released from cutaneous sensory nerve endings. CGRP marks intraepidermal nerve fibers (IENF). Apart from its role in pain transmission, CGRP is involved in sensitization of receptive endings. An increase in CGRP fibers in itchy skin conditions has been also observed (13). VIP-positive nerve fibers are found not only around sweat glands, root sheaths of hair follicles, and blood vessels but also in free nerve endings in papillary layer of the skin (14). Moreover, IENF can also display VIP immunoreactivity (15). VIP causes release of histamine from mast cells. In addition, this peptide intensifies acetylcholine-induced itch in atopic dermatitis patients (13). Increasing number of evidences shows that alterations in skin innervation in chronic pruritic skin conditions such as atopic dermatitis (16, 17), prurigo nodularis (18), or pruritic lesions of patients with psoriasis (19) may be a component of itch pathogenesis. Studies on vestibulodynia, a condition that also manifests sensory symptoms, have revealed changes in vulvar tissue innervation in affected patients (20).

Another form of itch, neuropathic itch, is related to disorders of central or peripheral nervous system. In peripheral neuropathy, pruritus sensation, often accompanied with burning and stinging sensation, arises due to the damage to nerve fibers (8). Small fiber neuropathy (SFN) results from the selective impairment of small nerve fibers (SNF) as follows: myelinated A-delta and unmyelinated C fibers. SFN has been associated with many medical conditions such as glucose dysmetabolism and metabolic syndrome, chronic infections (HIV, HCV), vitamin B12 deficiency, neurotoxic drug exposure, and paraneoplastic syndrome (21). What is more, alterations in skin innervation consistent with SFN have been observed in autoimmune diseases such as celiac disease (22), systemic lupus erythematosus (23), and primary Sjogren's syndrome (24). To diagnose SFN, the current guidelines recommend the use of skin biopsy to evaluate intraepidermal nerve fiber density (IENFD) as it is significantly decreased in affected patients. For this purpose, skin samples are stained immunohistochemically or by immunofluorescence with antibody against PGP 9.5, also known as ubiquitin carboxyl-terminal hydrolase-1 (UCH-L1), which marks nerve fibers (25).

Undoubtedly, there is increasing data and interest in investigations on the pathogenesis of pruritus. Because itch sensation is elicited on cutaneous free nerve endings of SNF, it was a matter of debate whether alterations in skin innervation were present in LS, like in other pruritic conditions. Changes in intraepidermal nerve fiber density seemed even more likely due to postulated autoimmune background of LS and association of SFN with autoimmune diseases.

Although a fragmentation of nerve fibers in the dermis in cases of LS was previously observed (26), alterations in the epidermal innervation by SNF have not been investigated so far. Therefore, the aim of our study was to compare IENF density in vulvar LS to control vulvar samples. These findings may contribute to understanding pathogenesis of itch sensation in LS. Beyond that, the expression of neuropeptides such as CGRP and VIP in vulvar LS was assessed to get further insight in the possible nerve alterations.

MATERIALS AND METHODS

Human skin biopsies

Of about 20 formalin-fixed paraffin-embedded (FFPE) specimens of skin biopsies of histologically confirmed vulvar LS were collected from the files of the Department of Pathomorphology, Jagiellonian University (Cracow, Poland). As a control group, surrounding healthy tissue from FFPE samples of 20 cases of benign and non-inflammatory vulvar lesions (including compound/dermal nevus, fibroepithelial polyp, cavernous hemangioma) from files of the Department of Pathomorphology, were analyzed. In LS group, patients aged between 34 and 76 years (mean 62 years) and ranged 29 – 77 years (mean 53 years) in control group (Table 1). It cannot be ascertained for sure that the patients had not been cured by whichever method before biopsy. Nevertheless, in all cases, in which we had more than one biopsy from the same patient, the earliest biopsy was included in the study. All cases were diagnosed between years 2006 and 2014. Because no established criteria of staging LS exist, we have arbitrarily established a staging system differentiating early, intermediate, and advanced stage of the disease according to the changes found in hematoxylin- and eosin-stained slides, based on the present literature on histological changes of the disease (27). Early stages of LS (n = 6) presented with prominent hyperkeratosis and a lymphocytic infiltration directly beneath the epidermis. Intermediate stages (n = 9) were characterized by further developed hyperkeratosis and beginning homogenization of collagen in the dermis as well as less prominent lymphocytic infiltrate in the dermis. Advanced stages (n = 5) showed progressing homogenization of collagen with flattening of the papillae in the stratum papillare of the dermis and epidermal atrophy.

Immunohistochemistry

Formalin-fixed paraffin-embedded skin biopsies were cut into thin sections (3 µm) for hematoxylin and eosin (H&E) staining and immunohistochemical analysis. For PGP 9.5 stain three serial sections were provided. All sections were prepared for immunohistochemical evaluation with the use of the following protocol.. After routine deparaffinization, rehydration, and blocking of endogenous peroxidase activity, freshly made 3% hydrogen peroxide in methanol was used for 20 min at room temperature. For antigen retrieval, using EDTA buffer pH 9.0 (only for CGRP for retrieval citrate buffer pH 6.0 was used), sections were microwaved for 20 min. Primary antibodies were used as follows: PGP 9.5 (clone 10A1, NovocastraTM, dilution 1:50, incubation 1 hour), VIP (clone H-6, Santa Cruz - sc-25347 mouse monoclonal antibody - 1:50 dilution, incubation 30 min, room temp.), CGRP (abcam ab54274 - rabbit polyclonal - 1:100 dilution, incubation 24 hours, overnight 4 – 8°C). After rinsing with Tris-borate buffer, the LabVision (Thermo Fisher Scientific, USA) detection system was used with 3,3'-diaminobenzidine (DAB) served as the chromogen, and the sections were finally counterstained with Mayer hematoxylin (30 s). To confirm the specificity of the primary antibody, positive and negative control test were performed, following manufacturing instructions. The negative control test included substitution of primary antibody with phosphate-buffered saline pH 7.4.

Intraepidermal nerve fiber estimation

Evaluation of IENFD was performed according to the guidelines of the European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy (25). Briefly, single IENF crossing the dermal-epidermal junction immunostained with PGP 9.5 was counted by two trained pathologists, under a bright-field microscope at high magnification (× 40) in always three serial cross-sections of one to four sections per biopsy. Each sample was assessed simultaneously on multi-head microscope by the observers to achieve unanimous consensus for the calculation. Single sections were used to calculate number of IENF for samples stained with CGRP and VIP.

Image and data analysis

Images were captured via video link to a microscope Nikon Eclipse Ci (× 40 objective) and scanned by the computer. The lengths of the dermal-epidermal junction were measured with ImageJ program for the calculation of exact length of dermal-epidermal junction and consequently exact linear epidermal innervation density (IENF/mm). Data were analyzed using SAS University Edition software. Determination of sample size was established according to the statistical method of power and sample size calculations. In order to examine the distribution of variables in the patients, the Shapiro-Wilk test was used. Using the parametric Student's t-test and non-parametric Mann-Whitney U-test, the clinical features of the study group and control group were compared where appropriate. One-way analysis of variance (ANOVA) was used to evaluate more than two groups of variables followed by Fisher's post hoc test where appropriate. Power analysis for one-way ANOVA was not performed. P < 0.05 indicated significance. Clinical features of the study patients are presented as mean values or number of cases. Figures were generated using pandas open source Phyton Data Analysis Library and Matplotlib, a python 2-D plotting library.

RESULTS

Histology and immunohistochemistry

PGP 9.5 immunoreactive fibers were detected throughout the dermis with fine fibers penetrating the epidermal layer in both controls and LS skin samples (Fig. 1). Mean IENFD in vulvar LS was 7.52 IENF/mm (values ranged from 2.69 to 20.15). In control group, mean IENF density was 12.25 IENF/mm (values ranged from 3.52 to 24.74) (Fig. 4). Mann-Whitney U test showed that a decrease of IENF density in vulvar LS compared to normal vulvar tissues can be stated with statistical certainty (P = 0.0049).

Table 1. Patients characteristics.

Fig. 1.Immunoreactivity for PGP 9.5 on cutaneous nerve fibers in vulvar skin. (A) Numerous intraepidermal nerve fibers (arrows) in early stage vulvar LS. (B) Cutaneous nerves mostly running parallel to flattened dermal-epidermal junction and single nerve fiber penetrating epidermis in advanced vulvat LS (arrow). (C) Small nerve fiber entering epidermis in normal vulvar skin (arrow).
e, epidermis; p, papillary dermis; A - C magnification × 400.

Fig. 2. Image analysis of intraepidermal nerve fiber density in normal vulvar tissue and vulvar LS immunostained with PGP 9.5 (A) and CGRP (B).

Correlation analysis showed a slight linear increase of IENF density with the age of the patients in both clinical and in control groups. One-way ANOVA results reveal gradual decrease of INEF density with the stage of disease. Post hoc test showed significant differences between early and advanced stage compared to control group. But, the analysis did not reveal differences between stage groups.

CGRP positive nerve fibers were present in the dermis as well as in the epidermis (Fig. 2). Mean IENFD were 7.35 IENF/mm in LS group (values ranged from 30.69 to 0.99) and 4.14 IENF/mm in control group (values ranged from 10.54 to 0.00) (Fig. 4). Unequivocal increase of number of CGRP positive nerve fibers in epidermis of lesional (LS) skin compared to normal vulvar skin was shown to be significant (Mann-Whitney U test, P = 0.03).

Fig. 3.Immunoreactivity for CGRP on cutaneous nerve fibers in vulvar skin. (A) (B) Fine nerve fibers (arrows) crossing dermal-epidermal junction in early stage vulvar LS. (C) Fine nerve fiber (arrow) penetrating epidermis in normal vulvar skin.
e, epidermis; p, papillary dermis; A - C magnification × 400.

Fig. 4.Immunoreactivity for VIP on cutaneous nerve fibers in vulvar skin. (A) Fine nerve fibers (arrows) crossing dermal-epidermal junction in intermediate stage vulvar LS. (B, C) Fine nerve fibers (arrow) penetrating epidermis in normal vulvar skin.
e, epidermis; p, papillary dermis; A - C magnification × 400.

Nerve fibers expressing VIP were observed mainly around sweat and sebaceous glands, root sheaths of hair follicle, and in papillary dermis and to a lesser extent in epidermis when compared to previous stains (Fig. 3). Mean IENFD was 5.44 IENF/mm in LS group (scores ranged from 13.20 to 0.00) and 4.54 IENF/mm in control group (scores ranged from 12.72 to 0.00). The obtained results did not differ significantly (Mann-Whitney U test, P = 0.18).

DISCUSSION

This study compared IENFD in vulvar LS to normal vulvar tissues using immunohistochemical markers for SNF: PGP 9.5, VIP, and CGRP. Modified in terms of tissue preservation and slide preparation, protocol for diagnosis of SFN was applied as it becomes standard procedure for establishing alterations in SNF (25). The obtained results demonstrate for the first time a decrease in the number of IENF in skin affected by LS. Moreover, despite a general decrease in the number of IENF, an increase in the number of itch-specific CGRP positive nerve fibers in lesional skin in LS has been observed. These findings point to damage to SNF in the disease process and suggest its role in pathogenesis of sensory symptoms of LS. The data on the relative gravity of symptoms have not been reported; therefore, one cannot specifically relate the subjective assessment of ailment to the obtained study results.

Small nerve fiber involvement has been shown in autoimmune diseases. Alterations of SNF consistent with diagnosis of SFN can be found in primary Sjogren's syndrome (24), systemic lupus erythematosus (23), and celiac disease (22). Studies showing decreasing IENFD in these chronic inflammatory autoimmune disorders might partially support the already favored theory of an autoimmune pathogenesis in LS. The strict criteria of SFN could not be applied here due to missing reference values for vulvar skin. One could speculate that not all patients in this study might be associated with an autoimmune form of LS. IgG autoantibodies against ECM-1 are usually present in only 74% of LS patients (2), and the number of patients with autoantibodies against BMZ is even lower (33%) (3). An analysis of the sera of the patients regarding autoantibodies against ECM-1 and BMZ could be useful to evaluate a possible association of decreased epidermal innervation with autoimmune mechanisms in the pathogenesis of LS.

SFN usually presents with symptoms of itch, pain, and burning, which might be caused by the damage to peripheral nerve fibers. These sensations are also typical for LS. According to the mechanism-based definition of pruritus, which differentiates itch sensation in normal nervous system triggered by peripheral and central mechanisms from sensation caused by changes in nerve fibers (neuropathic itch), we hypothesize that sensory symptoms in LS might be at least to some extent neuropathic in origin.

On the contrary, pruritic dermatoses such as atopic dermatitis (17), or pruritic lesions of patients with psoriasis (19, 28, 29), present with hyperinnervation of the lesional skin. The epidermal innervation is the result of balance between nerve elongation factors (e.g., nerve growth factor) and nerve repulsion factors (e.g., semaphorin 3A) (30). Increased innervation of epidermis by CGRP positive nerve fibers has been reported in psoriasis (31), chronic spontaneous urticarial (32), and hypertrophic burn scar (33). Scratching has been suggested to quickly (within 1 – 3 days) influence sensory nerve growth in the skin, and as a consequence, upregulate levels of CGRP as well as other neuropeptides via NGF-associated mechanisms (34). The hyperinnervation of epidermis by CGRP-positive nerve fibers in LS observed in our study might also be related to scratching in response to the itch sensation. Further studies on the expression of nerve growth factors and its receptors and inhibitors in lesional (LS) skin would probably shed more light onto the pathogenesis of discovered alterations of epidermal innervation. CGRP is also released from the central projections of dorsal root ganglia neurons where it may be involved in central sensitization in itch and pain pathways (34). Although the study on the molecular mechanisms of central sensitization in itch pathways is still in its early stage, such sensitization was demonstrated in patients with atopic dermatitis (9). The role of CGRP in pain transmission is still debated, and it may be possible that it becomes more important under conditions of abnormal pain processing and in inflammatory or neuropathic pain conditions. CGRP antagonists inhibited hyperalgesia induced by intraplantar injection of capsaicin and carrageenan in rats and reduced pain in neuropathic models (34). Moreover, evidence suggests that the sympathetic nervous system is involved in increasing dorsal root ganglia neuronal activity to promote pain. A recent study using cultured neurons of root ganglia showed that the molecular mechanism of this interaction, besides sensitization or upregulation of transient receptor potential vanilloid type 1, is also by reducing transient receptor potential vanilloid type 1 desensitization (35). To our knowledge, the role of central sensitization in pain and itch transmission in LS has not been investigated yet.

The intraepidermal innervation density decreases in the skin of the legs and also of the face with progressing age. But, abdominal skin shows no alteration of the intraepidermal innervation density, and mammary skin is even increasingly innervated (36). A study about the IENFD of vulvar skin in correlation with the age has not been performed yet. But, the results obtained here suggest, such as in mammary skin, that vulvar IENFD might progress with age.

In summary, this is the first report on immunohistochemical evaluation of IENFD in vulvar LS. Our data show a decrease in IENFD in LS-affected skin, which may explain to some extent the sensory symptoms of the disease. The obtained results may turn out to be useful for the development of novel antipruritic and anti-inflammatory therapeutic strategies.

Acknowledgements: The work was supported by Jagiellonian University grant No. K/ZDS/005739.

Conflict of interests: None declared.

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