Tattooing increases the number of Langerhans cells in skin: an immunocytochemical study.

Tattooing is an act of permanent marking of the skin with indelible patterns by pricking and inserting pigments. Langerhans cells (LCS) are dendritic cells normally present in suprabasal layers of the epidermis of the skin. To assess whether there were any effects caused by the tattooing on Langerhans cell population and cutaneous nerves, skin from affected areas (n = 15) was compared with controls (n = 10). Frozen sections were immunostained with antisera to S-100. No discernible change either in distribution or in number of Langerhans cells and nerves was seen upon comparison with control skin taken from different areas, but all of the specimens taken from affected areas had a significant increase in the number of Langerhans cells (p < 0.001) even after several years of tattooing with no change in the cutaneous nerves. Thus, the study shows persistent stimulation of Langerhans cell population in tattooed skin.

Ontogeny of nerves and neuropeptides in skin of rat: an immunocytochemical study.

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.

Symptoms of notalgia paresthetica may be explained by increased dermal innervation.

Notalgia paresthetica is a sensory neuropathy characterized by infrascapular pruritus, burning pain, hyperalgesia, or tenderness. To assess whether the symptoms may be caused by alterations in the cutaneous innervation, skin from the affected area of patients (n = 5) was compared with controls (n = 10) comprising the contralateral unaffected area from the same patients and site-matched biopsies of normals, using immunohistochemistry. Frozen sections were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide with tyrosine, and to the general neural marker PGP 9.5 and the glial marker S-100 to show the overall innervation and glial cells, respectively. No discernible change in the distribution of neuropeptide-immunoreactive axons was found, but all of the specimens from the affected areas had a significant increase in the number of intradermal PGP 9.5-immunoreactive nerve fibers compared with unaffected areas from the same patients and normal controls. Epidermal dendritic cells immunoreactive for S-100, possibly Langerhans cells, were substantially increased. It is concluded that there is an increase in the sensory epidermal innervation in the affected skin areas in notalgia paresthetica, which could contribute to the symptoms, and that neural immunohistochemistry of skin biopsies could be helpful in the diagnosis of the disease.

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.

Reinnervation and neuropeptides in mouse skin flaps.

Degeneration and regeneration of nerves in skin flaps has so far been studied mainly by classical staining methods and there is little information on neuropeptide involvement. Therefore, we have investigated immunocytochemically the temporal course of reinnervation of neuropeptide appearance in skin flaps of mice. Fibres immunoreactive for a general neural marker protein gene product 9.5 (PGP 9.5), and for the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) disappeared by 24 h in the flap and surrounding skin. By the 7th day, PGP 9.5-immunoreactive nerves were seen close to the pedicle. By 15 days, they were seen within the body of the flap, clustered focally without specific organisation in greater density than in control skin. Many were also immunoreactive for CGRP and substance P. Neural immunoreactivity for VIP and NPY appeared later (day 30). Thus, regrowing nerves appear initially to be denser than normal innervation. The early appearance of apparently hypertrophic sensory neuropeptide-containing (CGRP and substance P) fibres may have a role in trophic, regulatory and reparative processes.

Time-related decrease of substance P and CGRP in central and peripheral projections of sensory neurones in Mycobacterium leprae infected nude mice: a model for lepromatous leprosy in man.

We have previously shown the depletion of cutaneous calcitonin gene-related peptide (CGRP)- and substance P-containing nerves in human leprosy. The aims of this study were to investigate the temporal effects of leprosy on nerves in skin and spinal cord. Tissues were taken from nude mice, 6 and 12 months after inoculation of Mycobacterium leprae into the hind footpads, and from age-matched controls. Sections were immunostained with antisera to substance P or CGRP. After 6 months of infection, substance P- and CGRP-immunoreactive nerves were reduced in skin from all body areas; by 12 months, the reduction was substantially greater. In the spinal cord, sensory fibres immunoreactive for substance P had decreased compared with controls at 6 and 12 months [by 60 per cent (0.022 mm2) and 80 per cent (0.048 mm2), respectively, P less than 0.001], as with CGRP [30 per cent (0.018 mm2) (P less than 0.02) and 40 per cent (0.028 mm2) (P less than 0.01), respectively]. CGRP immunoreactivity was completely absent in motor neurones after 12 months of infection. Loss of CGRP- and substance P-immunoreactive fibres in skin and spinal cord, and CGRP in motor neurones is in accord with impaired pain sensation and muscle weakness in leprosy.

Early increase in CGRP- and VIP-immunoreactive nerves in the skin of streptozotocin-induced diabetic rats.

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.

Depletion of cutaneous nerves and neuropeptides in diabetes mellitus: an immunocytochemical study.

Immunocytochemistry for the general neuronal marker protein gene product 9.5 and four neuropeptides (calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide and neuropeptide Y) was performed on 20 skin biopsy specimens from 19 diabetic patients, age range 20-75 years, 17 Type 2 (non-insulin-dependent) and 3 Type 1 (insulin-dependent). Fifteen specimens were from the lower limb, 3 from the upper limb and 2 from the abdominal wall. Seven subjects had lower limb neurophysiological tests. All but one specimen showed reduced protein gene product 9.5 and neuropeptide immunoreactivity. Reduced protein gene product 9.5 and neuropeptide immunoreactivity was found in specimens taken from the abdominal wall and hand as well as those from the leg, and also in specimens from patients undergoing amputation for peripheral vascular disease. In general, the greater the number of abnormal neurophysiological tests, the greater the extent of neuronal abnormalities. Three patients with normal tests had abnormalities of dermal innervation. While these changes are also found in other axonal neuropathies, in the absence of other causes of peripheral nerve disease and of macrovascular disease, immunocytochemistry of skin biopsies may have a role in the assessment of diabetic neuropathy and its response to treatment.

Changes in nerves and neuropeptides in skin from 100 leprosy patients investigated by immunocytochemistry.

The cutaneous innervation is now known to contain neuropeptides including substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory nerves, and vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), principally in autonomic nerves. Skin biopsies from 100 leprosy patients and equivalent areas from 50 non-leprosy controls were fixed in p-benzoquinone solution for immunofluorescence staining and in Bouin's fluid for classification of leprosy type. Antisera to the neural markers, neurofilaments, and protein gene product 9.5 (PGP 9.5), and to neuropeptides were used. Cutaneous nerves and nerve endings immunoreactive for neuropeptides, neurofilaments, and PGP 9.5 were seen in all non-leprous control cases. In leprosy, PGP 9.5- and neurofilament-immunoreactive nerve fibres were seen in all 14 cases of the indeterminate (early) type and in the majority (33/43) of lepromatous cases, but in a smaller proportion (15/43) of tuberculoid cases. Neuropeptide immunoreactivity was seen in only 2/14 of the indeterminate leprosy specimens and was completely absent in other types. This early disappearance may be of diagnostic significance. Thus, cutaneous sensory and autonomic dysfunctions in leprosy are well reflected by changes in nerve fibres and neuropeptides.