Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation

Background A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease. Objective To obtain simultaneously microscopic images and spectra of collagen and elastin fibres in ex vivo skin tissues (normal skin and hypertrophic scar) using a nonlinear spectral imaging method, and to compare the morphological structure and spectral characteristics of collagen and elastin fibres in hypertrophic scar tissues with those of normal skin, to determine whether this approach has potential for in vivo assessment of the pathophysiology of human hypertrophic scars and for monitoring treatment responses as well as for tracking the process of development of hypertrophic scars in clinic. Methods Ex vivo human skin specimens obtained from six patients aged from 10 to 50 years old who were undergoing skin plastic surgery were examined. Five patients had hypertrophic scar lesions and one patient had no scar lesion before we obtained his skin specimen. A total of 30 tissue section samples of 30 μm thickness were analysed by the use of a nonlinear spectral imaging system consisting of a femtosecond excitation light source, a high‐throughput scanning inverted microscope, and a spectral imaging detection system. The high‐contrast and high‐resolution second harmonic generation (SHG) images of collagen and two‐photon excited fluorescence (TPEF) images of elastin fibres in hypertrophic scar tissues and normal skin were acquired using the extracting channel tool of the system. The emission spectra were analysed using the image‐guided spectral analysis method. The depth‐dependent decay constant of the SHG signal and the image texture characteristics of hypertrophic scar tissue and normal skin were used to quantitatively assess the amount, distribution and orientation of their collagen and elastin components. Results Our experiments and data analyses demonstrated apparent differences between hypertrophic scar tissue and normal skin in terms of their morphological structure and the spectral characteristics of collagen and elastin fibres. These differences can potentially be used to distinguish hypertrophic scar tissues from normal skin and to evaluate treatment responses. Conclusions All the measurements were performed in backscattering geometry and demonstrated that nonlinear spectral imaging has the ability to differentiate hypertrophic scar tissue from normal skin based on noninvasive SHG imaging, and TPEF imaging revealed the microstructure and spectral features of collagenand elastin fibres. With the advances in spectral imaging apparatus miniaturization, we have good reason to believe that this approach can become a valuable tool for the in vivo pathophysiology study of human skin hypertrophic scars and for assessing the treatment responses of this disfiguring disease in clinic. It can also be used to track the development of hypertrophic scars and to study wound healing processes in a noninvasive fashion without biopsy, fixation, sectioning and the use of exogenous dyes or stains.     

Neuropeptides in the skin of patients with atopic dermatitis

There is increasing evidence that neuropeptides may be involved in the pathogenesis of atopic dermatitis (AD). This study examines whether neuropeptide distribution in the skin of patients with AD differs from normal controls. The distribution and density of several neuro-peptides were examined in lesional and non-lesional skin of AD patients (n= 5) and in normal controls (n= 4) using indirect immunofluorescence and image analysis. Cholinergic innervation was studied using cholinesterase histochemistry. Staining with the general neuronal marker protein gene product 9·5 showed a subepidermal network of nerves with fibres penetrating the epidermis, and nerves around blood vessels, sweat glands and hair follicles. Image analysis of nerves around sweat glands showed a significantly higher nerve density in non-lesional compared with both normal controls and lesional skin (P < 0·05); lesional compared with control skin showed no significant difference. In the epidermis the density of nerves was not significantly greater in non-lesional compared with lesional skin and controls. Calcitonin gene-related peptide immunoreactivity was similar in all subjects except in three of the AD patients, where more nerves appeared to penetrate the epidermis. Substance P immunoreactivity in the papillary dermis was seen in all AD patients hut no controls. Vasoactive intestinal polypeptide and neuropeptide Y staining were similar in all groups. Acetyleholinesterase-positive nerves were found around sweat glands in all subjects, the staining being greatest in non-lesional and least in lesional skin. Occasional nerves were seen in the papillary dermis in lesional skin of two out of the four patients. We have demonstrated quantitative differences in nerve growth in clinically normal skin of AD patients, and altered cutaneous neuropeptide expression in these patients which may contribute to the pathogenesis of AD.     

cis-Urocanic acid stimulates neuropeptide release from peripheral sensory nerves.

Previous studies using an antibody to cis-urocanic acid and mast-cell-depleted mice implicated both cis-urocanic acid and mast cells in the mechanisms by which ultraviolet B light suppresses systemic contact hypersensitivity responses in mice. In the absence of a direct stimulatory effect of cis-urocanic acid on connective tissue mast cells, an indirect association was investigated. A blister induced in the rat hind footpad was used to examine the effects of slowly perfused cis-urocanic acid on cutaneous blood flow. cis-Urocanic acid but not trans-urocanic acid increased microvascular flow by a mechanism largely dependent on the combined activity of the neuropeptides, substance P and calcitonin gene-related peptide. Perfusion of cis-urocanic acid over the base of blisters induced in sensory-neuropeptide-depleted rats did not have any stimulatory effect above that seen with perfusion of cis-urocanic acid together with neuropeptide receptor antagonists in control rats. There was a small direct effect of cis-urocanic acid on microvascular blood flow. As both substance P and calcitonin gene-related peptide could directly degranulate connective tissue mast cells, this study suggests that cis-urocanic acid indirectly activates mast cells via its effects on peripheral terminals of unmyelinated primary afferent sensory nerves. cis-Urocanic-acid-induced neuropeptides may also contribute to ultraviolet-B-induced cutaneous inflammation and alterations to Langerhans cell activity.     

Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring

Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix‐associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type‐I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three‐dimensional type‐I collagen lattices. Inhibition of Rho‐associated protein kinase activity significantly attenuated the effects of matrix‐associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix‐associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho‐associated protein kinase‐dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.     

Reduced collagenase gene expression in fibroblasts from hypertrophic scar tissue

Summary The major histopathological feature of hypertrophic scar lesions is fibrosis. characterized by excessive accumulation of collagen. The purpose of this study was to determine if there is not only increased expression of collagen but also decreased expression of collagenase in hypertrophic scar fibroblasts. We compared the expression of mRNA for of α₁ (I) and α₁ (III) collagen, and collagenase in cultured fibroblasis from different portions of hypertrophic scars and normal dermis. the hypertrophic scar fibroblasts. increased levels of α₁ (I) and α₁ (III) collagen mRNAs were observed in fibroblasts from the edge and outside of scar tissue, while normal levels were noted in fibroblasts from the centre of this tissue. In contrast. decreased levels of collagenase mRNA were found in the hypertrophic scar fibroblasts. The reductions were centre (25% of the control) greater than the edge (43% of the control) greater than the outside (84% of the control). The changes in the collagenase mRNA levels of the hypertrophic scar fibroblasts correlated well with decreased collagenolytic activity as determined by the degradation rate of fluorescent isothiocyanate-labelled type I collagen in fibroblast culture supernatant. These results suggest that decreased expression of collagenase in hypertrophic scar fibroblasts may be one possible cause for the excessive accumulation of collagen in the skin lesions of hypertrophic scars.     

Ontogeny of nerves and neuropeptides in skin of rat: An immunocytochemical study

Abstract The sequence of maturation of nerves and appearance of neuropeptides was investigated in skin from fetal and neonatal rats by immunocytochemistry using antisera to protein gene product 9.5, substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Immunoreactivity for PGP 9.5 appeared on fetal day 16 in face and nose, somewhat later (fetal day 19) in paws and tail. The sensory neuropeptides, CGRP/substance P (fetal day 19 and postnatal day 1, respectively) appeared earlier than the autonomic peptides VIP and NPY (postnatal day 7). Thus, the study shows that neuropeptides do not appear simultaneously with nerves and that the development is rostrocaudal.     

Depletion of cutaneous peptidergic innervation in HIV-associated xerosis

Severe xerosis occurs in approximately 20% of human immunodeficiency virus seropositive patients. Changes in cutaneous innervation have been found in various inflammatory skin diseases and in xerotic skin in familial amyloid. We have therefore carried out a quantitative examination of the cutaneous peptidergic innervation in human immunodeficiency virus-associated xerosis. Immunohistochemistry and image analysis quantitation were used to compare total cutaneous innervation (protein gene product 9.5), calcitonin gene-related peptide, substance P, and vasoactive intestinal peptide peptidergic fibers, at two sites in the skin of human immunodeficiency virus-associated xerosis patients (upper arm, n = 12; upper leg, n = 11) and site-matched seronegative controls (upper arm, n = 10; upper leg, n = 10). Measurement of lengths of fibers of each type was carried out for each subject in the epidermis and papillary dermis, and around the sweat glands. Immunostained mast cells in these areas were counted. Epidermal integrity and maturation were assessed by immunostaining for involucrin. There were significant (Mann-Whitney U test; p < 0.02) decreases in total lengths of protein gene product 9.5 fibers in both epidermis/papillary dermis and sweat gland fields; of calcitonin gene-related peptide innervation in the epidermis/papillary dermis; and of substance P innervation of the sweat glands. There were no differences in the distribution of mast cells, or in the epidermal expression of involucrin. Depletion of the calcitonin gene-related peptide innervation may affect the nutrient blood supply of the upper dermis, and the integrity and function of basal epidermis and Langerhans cells. Diminished substance P innervation of the sweat glands may affect their secretory activity. Both of these changes may be implicated in the development of xerosis.     

Occurrence and distribution of neuropeptides in the human skin. An immunocytochemical and immunochemical study on normal skin and blister fluid from inflamed skin

The content and distribution of substance P (SP), somatostatin, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in human skin were investigated. Radioimmunoassay was performed on pooled tissue samples from several regions (fingers, toes, axillas and thighs) and on tissue fluid from spontaneous blisters on inflamed skin. Immunocytochemical localization showed all peptides examined except somatostatin to be present in nerve fibers. Nerve fibers storing SP and CGRP, which were found to coexist, were mostly present as free nerve endings in the superficial part of dermis and in epidermis. SP/CGRP fibers were most abundant in fingers and toes. VIP fibers and NPY fibers were localized in the deeper parts of dermis around blood vessels and acini of sweat glands. Also fibers containing these neuropeptides were most common in fingertips and toes. VIP occurred in relatively high amounts also in skin from axilla whereas NPY in this region was below detection limit. Immunoreactive somatostatin was found in low concentrations in tissue extracts and was not present in amounts sufficient for reliable immunostaining. Fluid from spontaneous blisters on inflamed skin contained detectable amounts of all neuropeptides.     

Folliculosebaceous Cystic Hamartoma with a Neural Component: An Immunohistochemical Study

We present a case of follicular cystic hamartoma, a distinctive cutaneous malformation characterized by marked overgrowth of folliculosebaceous units accompanied by appreciable mesenchymal alterations, including fibroplasia, increased vascular components, and numerous adipocytes. A conspicuous feature of our case is an aggregation of thick trespassing nerve bundles in the deep portion of the neoplasm. An immunohistochemical study revealed the nerve bundles were immunoreactive for the general neuronal marker protein gene product 9.5. The nerves, however, stained negatively with antibodies against neuropeptides such as calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, and neuropeptide Y, all of which are known to be contained in normal cutaneous nerves. The presence of these aberrant nerve bundles devoid of the normal expression of neuropeptides supports the concept that folliculosebaceous cystic hamartoma is a true, tumor-like malformation characterized by abnormal overgrowth of normal components of the skin.     

VITAMIN E ADDED SILICONE GEL SHEETS FOR TREATMENT OF HYPERTROPHIC SCARS AND KELOIDS

Background. Trauma of various origins can induce a connective tissue disorder that leads to keloids. This condition has yet not been clearly distinguished from scars and hypertrophic scars. Only electronmicroscopic and biochemical data can help to do this. Among some more or less therapeutic approaches, the use of silicon (polydimethylsiloxane) plates, wrapped on the keloid surface, has been reported effective by some authors. These authors also demonstrated that it is not the mechanical pressure that is the mechanism of action, but a direct action on fibroblasts and a hyperhydration of subcutaneous tissue. The authors of this study conceived that the silicon plate may be able to improve the transdermal penetration of a compound such as vitamin E. This vitamin is capable of preserving some important morphologic and functional features of biological membranes by means of its phytilside chain of the molecule acting as a stabilizer of lysosomal membranes. Materials and Methods. Eighty patients of both sexes, aged between 18 and 63 years, who had hypertrophic scars and keloids, were admitted to the trial. The patients were randomized to two groups in a simple-blinded study. Group A: Forty patients whose scars have been covered with silicon plates with added vitamin E. Group B: Forty patients treated with simple silicone gel sheets. No pressure bandages were used, only tape fixing the sheet for 10 hours overnight. The trial lasted for 2 months. The results were recorded at 4 and 8 weeks, evaluating the improvement according to a Scott-Husskinson scale. For objective assessment photos were taken. The results were analyzed by the chi-square test. Results. At the end of the first month, group A had improved by more than 50% in 85% of cases, whereas the improvement in group B was 55% (P < 0.01). At the end of the second month, 95% of patients in group A had improved by 50%, whereas 75% had improved by 50% in group B (P < 0.05). Conclusions. Vitamin E added to the silicon plate scored better than the simple silicon plate at the end of both periods. We have reported the successful combined action of vitamin E and silicone gel sheets in scar treatment, especially in the short-term prophylaxis of hypertrophic scars or keloids.     

Keloids and hypertrophic scars

Keloids and hypertrophic scars occur in predisposed individuals following trauma, inflammation, surgery, or burns and occasionally they occur spontaneously. These fibrous growths are abundant depositions of collagen and glycoprotein. Early reports often included both keloids and hypertrophic scars from various anatomic sites. Such reports add confusion to a poorly understood wound response. Later reports distinguish keloids from hypertrophic scars in terms of extension beyond the original wound and clinical course. Hypertrophie scars typically remain within the confines of the original wounds and these raised scars often spontaneously regress. Hypertrophic scars occur soon after the antecedent trauma or inflammation. In general, the hypertrophic scar remains within the limit of the wound or inflammation and the lesion may increase in size or regress over months to years. Keloids occur in predisposed individuals and the enlarging lesions extend beyond the original wound and rarely regress.¹ Individuals with severely disfiguring multiple keloids are more likely to have a family history of keloids, suggesting a genetic predisposition for this abnormal wound response. The cause of this abnormal wound response is unknown, but abnormal cellular responses may account for the abundant connective tissue deposition. These fibrous growths typically generate significant disfigurement and unwanted symptoms, such as pruritus or pain. Multiple treatment approaches have been reported and responses are highly variable. Therapy selection should be carefully matched to the individual and the abnormal wound response to obtain the best possible result.     

Skin nerve fibres and their contacts with mast cells in patients with palmoplantar pustulosis

Abstract Patients with palmoplantar pustulosis (PPP) frequently report that stress worsens their condition. A study was therefore made of the distribution and number of nerve fibres positive for protein gene product (PGP) 9.5 (a general nerve marker) and nerve fibres with substance P- and calcitonin gene-related peptide-like immunoreactivity in involved skin from patients with PPP and in skin from healthy controls. The number of mast cells in the papillary dermis was larger (P = 0.0003) in lesional palmar PPP skin than in control skin, and the number of contacts between mast cells and nerve fibres was significantly larger (P = 0.02) in PPP skin than in control skin. Image analysis of the nerve fibres around the sweat glands showed that the positively stained area as a percentage of the total area of the sweat gland (coil + surrounding nerves) was significantly lower in PPP skin (P = 0.0006). Furthermore, the nerves seemed to be fragmented. Neutrophils within and below the pustules and in the papillary dermis showed positive substance P staining. The increased number of contacts between nerves and mast cells in PPP skin and the intense substance P-like immunoreactivity of the neutrophils indicate that neuromediation may influence the inflammation in PPP, whereas the destruction of the nerve fibres around the sweat glands might be a result of the inflammation. Received: 8 October 1999 / Revised: 25 January 2000 / Accepted: 27 January 2000     

Neuropeptides in cutaneous neurofibromas of Von Recklinghausen''s disease

The occurrence of neuropeptides was studied in neurofibromas of von Recklinghausen's disease by indirect immunofluorescence. All non-plexiform cutaneous neurofibromas contained abundant vasoactive intestinal polypeptide, peptide histidine-isoleucine and calcitonin gene-related peptide immunoreactive nerves. The nerves were small and unmyelinated. Neuropeptides might be responsible for itch that occurs especially in small cutaneous neurofibromas. Neuropeptides are also suggested to act as modulators and/or trophic factors for neurofibroma growth.     

Pulsed dye laser versus long-pulsed Nd:YAG laser in the treatment of hypertrophic scars and keloid: A comparative randomized split-scar trial

Introduction: Keloids and hypertrophic scars are benign fibrous growths that occur after trauma or wounding of the skin and present a major therapeutic problem. Objective: The purpose of this study is to evaluate and compare the effectiveness of pulsed dye laser (PDL) versus Nd:YAG laser in hypertrophic scar and keloid. Methods: Twenty patients with hypertrophic scars and keloid were included in this prospective, randomized, split-scar study. Half of each scar was randomized to treatment with a 595-nm PDL and the contralateral half with the 1064-nm Nd:YAG. Each patient received 6 laser treatment sessions at 1-month intervals. The scars were evaluated at baseline and one month after the last laser session using the Vancouver scar scale (VSS). Results: One month after the last laser treatment, final total VSS analysis of treated sites by PDL and long-pulsed Nd:YAG laser revealed significant improvements (p < 0.001), whereas the average percentage of improvement in the total VSS was 55.14% for PDL and 65.44% for Nd:YAG laser. However, there were no statistically significant differences between PDL- and long-pulsed Nd:YAG laser-treated sites for total VSS (p = 0.074). Limitations: This was a single-center non-controlled trial, which included a small number of patients and subjective outcome measures. Conclusion: PDL and long-pulsed Nd:YAG laser treatments for keloid and hypertrophic scar provide significant improvement with insignificant difference between both modalities.     

Oleanolic acid inhibits hypertrophic scarring in the rabbit ear model

Background. Hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, results from an overproduction of collagen and excessive deposition of extracellular matrix. Although treatment with surgical excision or steroid hormones can modify the symptoms, numerous treatment‐related complications have been described. Aim. To investigate the effects of oleanolic acid (OA), a naturally occurring triterpenoid, on hypertrophic scarring in a rabbit ear model. Methods. A rabbit ear model of hypertrophic scarring was used, with wounds produced with a biopsy punch. Oleanolic acid (2.5%, 5% and 10%) was applied once daily to the scars for 22 days. On postoperative day 28, the scars were excised, and the tissue used for histological examination and assays of the levels of collagens I and III, matrix metalloproteinase (MMP)‐1 and transforming growth factor (TGF)‐β₁. The scar elevation index (SEI) was also determined. Results. Treatment with different concentrations of oleanolic acid (OA) for 22 days significantly inhibited hypertrophic scarring in rabbit ear tissue. Levels of TGF‐β₁, collagen I and collagen III were significantly decreased and levels of MMP‐1 significantly increased in the scar tissue. SEI was also significantly reduced. Histological findings showed significant amelioration of the scar tissue. Conclusions. OA suppresses hypertrophic scarring in the rabbit ear model and may be an effective cure for human hypertrophic scarring.     

Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance

The skin as a barrier and immune organ is exposed to omnipresent environmental challenges such as irradiation or chemical and biologic hazards. Neuropeptides released from cutaneous nerves or skin and immune cells in response to noxious stimuli are mandatory for a fine-tuned regulation of cutaneous immune responses and tissue maintenance and repair. They initialize host immune responses, but are equally important for counter regulation of proinflammatory events. Interaction of the nervous and immune systems occurs both locally - at the level of neurogenic inflammation and immunocyte activation - and centrally - by controlling inflammatory pathways such as mononuclear activation or lymphocyte cytokine secretion. Consequently, a deregulated neurogenic immune control results in disease manifestation and frequently accompanies chronic development of cutaneous disorders. The current understanding, therapeutic options, and open questions of the role that neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide, neuropeptide Y, or others play in these events are discussed. Progress in this field will likely result in novel therapies for the management of diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm.     

The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography

In this study radioimmunoassay was used to determine neuropeptide levels in extracts from 17 differing anatomical regions of human skin. Marked regional variations of neuropeptide content for human skin were found and these variations are likely to reflect true physiological functions for the neuropeptides studied. In general the tachykinins, substance P (SP), neurokinin A (NKLA) and calcitonin gene-related peptide (CGRP) were found in highest concentrations in regions of skin with the greatest tactile sensation. By contrast, highest concentrations of vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) were found in axillary skin, where they probably play a part in axillary eccrine sweat production. Neurotensin was not found in any of the skin areas sampled, suggesting that it is relatively unimportant in human physiological skin control. Reverse-phase high-performance liquid chromatography (rpHPLC) was used to verify the results of radioimmunoassay. Both SP and NKA occurred in several regions in both their reduced and oxidized forms, as well as displaying molecular heterogeneity. CGRP occurred as one molecular species, this being α-CGRP, suggesting that this is the predominant molecular form in human skin. Likewise, both VIP and PHM displayed molecular homogeneity in the regions investigated by rpHPLC.     

Human hypertrophic and keloid scar models: principles,limitations and future challenges from a tissue engineering perspective

Most cutaneous wounds heal with scar formation. Ideally, an inconspicuous normotrophic scar is formed, but an abnormal scar (hypertrophic scar or keloid) can also develop. A major challenge to scientists and physicians is to prevent adverse scar formation after severe trauma (e.g. burn injury) and understand why some individuals will form adverse scars even after relatively minor injury. Currently, many different models exist to study scar formation, ranging from simple monolayer cell culture to 3D tissue‐engineered models even to humanized mouse models. Currently, these high‐/medium‐throughput test models avoid the main questions referring to why an adverse scar forms instead of a normotrophic scar and what causes a hypertrophic scar to form rather than a keloid scar and also, how is the genetic predisposition of the individual and the immune system involved. This information is essential if we are to identify new drug targets and develop optimal strategies in the future to prevent adverse scar formation. This viewpoint review summarizes the progress on in vitro and animal scar models, stresses the limitations in the current models and identifies the future challenges if scar‐free healing is to be achieved in the future.     

Neuropeptides and general neuronal marker in psoriasis an immunohistochemical study

Nerve fibres immunoreactive to antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were increased in lesional psoriatic skin when assessed semi-quantitatively. Biopsies from psoriatic plaques on the arm were studied in 13 patients and compared with biopsies from non-lesional areas (in three of the same psoriatic subjects) and from normal skin in seven non-psomtic controls. Immunohistochemical methods were used on cryocut skin sections to demonstrate the neuropeptides SP, VIP, calcitonin gene-related peptide and neuropeptide Y, and the general neuronal marker protein gene product (PGP) 9.5. The immunofluorescence was examined by semiquantitative and, for PGP 9.5, by quantitative methods. VIP reactive nerve fibres were increased at areas of eccrine sweat glands throughout the dermis, at the dermo-epidermal junction, and in the epidermis, in psoriasis lesional skin. SP reactive nerve fibres were increased at the dermo-epidermal junction, where the nerves ran parallel with and perpendicularly through the junction. PGP 9.5 reactive nerve fibres showed an increase at the dermo-epidermal junction, in the papillary dermis, and at the eccrine sweat glands in lesional psoriatic skin but not in non-lesional, or in control skin. These findings support the hypothesis that neuropeptides may be involved in the pathogenesis of psoriasis.