Human Digital Skin Research Articles

Verification and characterisation of human digital Ruffini's sensory corpuscles.

Ruffini's corpuscles are present as long fusiform encapsulated sensory structures in different tissues including the skin. Although physiological analyses strongly suggest their existence in glabrous digital skin, such localisation remains unconfirmed. Here, we have investigated the occurrence of typical Ruffini's corpuscles in 372 sections of human digital skin obtained from 186 subjects of both sexes and different ages (19-92years). S100 protein, neuron-specific enolase and neurofilament proteins were detected, and the basic immunohistochemical profile of these corpuscles was analysed. Fewer than 0.3 Ruffini's corpuscles/mm2 were detected, with density distribution across the fingers being F4>F3>F2>F1>F5 and absolute values being F2>F1>F3>F4>F5. Axons displayed neuron-specific enolase immunoreactivity, glial cells forming the core contained S100 protein, and the capsule was positive for CD34 but not Glut1, demonstrating an endoneurial origin. Present results demonstrate the existence of Ruffini's corpuscles in human glabrous digital skin at very low densities. Moreover, the identified Ruffini's corpuscles share the basic immunohistochemical characteristics of other dermal sensory corpuscles.

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles andlanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry toinvestigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20+ , chromogranin A+ and synaptophisin+ ) Merkel cells. Most of them were in close contact with Piezo2- nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2+ , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans.

The collagenic structure of human digital skin seen by scanning electron microscopy after Ohtani maceration technique

We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal papillae. At high magnification, we observed the spatial arrangement of the collagen fibers constituting the top of the dermal papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes.

Neuronal structural proteins, transmitters, transmitter enzymes and neuropeptides in human Meissner's corpuscles: a reappraisal using immunohistochemistry.

The innervation of Meissner's corpuscles (Mc) is complex, consisting of different types of sensory nerve fibers. We investigated the neurochemistry of Mc in human digital skin by indirect immunofluorescence, using a wide panel of both general neuronal as well as neurotransmitter-related molecules. Structural proteins (protein gene product 9.5, neuron-specific enolase, neurofilament) were found to consistently label the entire neuronal component of Mc. Immunoreactivity for gamma-melanocyte stimulating hormone was detected in the large diameter fibers running spirally within the corpuscles, while a number of peptide transmitters (substance P, calcitonin gene-related peptide, neurokinin A, galanin, somatostatin) were found in the thin unmyelinated fibers in both intra- and extracorpuscular locations.

Merkel complexes of human digital skin: Three‐dimensional imaging with confocal laser microscopy and double immunofluorescence

Merkel complexes of human digital skin: Three‐dimensional imaging with confocal laser microscopy and double immunofluorescence

Merkel complexes of human digital skin: Three-dimensional imaging with confocal laser microscopy and double immunofluorescence

Three-dimensional (3-D) reconstruction of images provided by confocal scanning laser microscopy (CSLM) is a powerful tool in a morpho-functional approach to cutaneous innervation studies. To investigate mechanoreceptors in the hand, a study of Merkel complexes was performed in human finger. A double fluorescent-conjugated immunolabeling with antibodies against neurofilament (NF 200) and cytokeratin (CK 20) on floating, thick cutaneous samples (80 to 100 microm), was used. After acquisition of serial optical planes by CSLM, reconstruction was performed with 3-D reconstruction software tools. Merkel cells were clearly labeled with CK 20, whereas nerve components were only NF 200 reactive. The cells, localized on the basal lamina of the epidermis, were usually arranged in clusters of five to eight cells. Each cell was connected to a nerve process ramification originating from a unique fiber. Quantitative data, compiled from a sample of 25 Merkel complexes, gave a mean cell diameter of 13 +/- 1 microm and a mean nerve fiber size of 3 +/- 1 microm. Surface measurements were done on a single reconstructed cluster with a mean and standard error which only refers to the optical 3-D resolution. It gives a surface of 12 +/- 1 microm2 for the contact zone between cell and nerve fiber and a cluster area of about 500 microm2. The great precision of reconstructed images provides a detailed analysis of spatial relationships between abutting nerve fibers and Merkel cells. Data interpretation is improved with complementary ultrastructural and physiological studies results, and this allows an accurate investigation of cutaneous sensory endings.

Study of human cutaneous sensory corpuscles using double immunolabelling and confocal laser scanning microscopy.

The main constituents of sensory corpuscles, i.e., the central axon, the periaxonic Schwann-related, cells, and the perineurial-related cells, can be identified light microscopically by simple immunohistochemistry using specific antibodies. This paper demonstrates the usefulness of double immunolabelling for light and confocal laser-scanning microscopy (CLSM) in the study of human cutaneous sensory corpuscles. Antibodies directed against neurofilament proteins (NFPs) and S-100 protein were used to label the central axon and the lamellar cells of Meissner corpuscles or the inner-core lamellae of digital cutaneous Pacinian corpuscles, respectively. Samples were obtained from subjects with normal sensitivity and from patients with paresthesia or absence of clinical sensitivity. Single and double immunolabelling was performed, and the sections were studied by light or CLSM microscopy. Double immunolabelling was effective for simultaneous observation of the central axon (NFP-positive) and periaxonic Schwann-related (S-100 protein-positive) cells in sensory corpuscles from normal human digital skin. The images that were obtained with both methods were comparable, but the axonic profiles were sharper with diaminobenzidine (DAB) used as a chromogen rather than with Texas-red used as a fluorochrome. Nevertheless, the ability to manipulate the focal plane by using CLSM permits one to better analyze the intracorpuscular relationships of the axon. Double immunolabelling in sensory corpuscles from the skin of patients with nerve compression showed the presence of a central axon in the corpuscles, whereas it was absent in the sensory corpuscles of clinically denervated skin. Double immunolabelling is a useful method with which to analyze simultaneously two of the corpuscular constituents, and it may be used in the study of denervated and reinnervated sensory corpuscles.

β-Amyloid precursor protein in human digital skin

The occurrence and distribution of β-amyloid precursor protein (βAPP) and of β-amyloid peptide (β/A4) was investigated using immunoblotting and immunohistochemical techniques in the digital skin of healthy adult subjects. βAPP-like proteic bands with apparent molecular masses between 55–60 kDa, 100–125 kDa (corresponding to the full-length βAPP isoforms), 145–150 kDa, and 200 kDa were found in pellets and supernatants of whole skin and dermis. The same proteins, except that of ∼200 kDa, were also found in pellets from the epidermis, whereas epidermic supernatants were unreactive. β/A4 was not found by immunoblotting. Light microscope immunohistochemistry showed βAPP immunoreactivity (IR) in: (a) dermal nerves; (b) lamellar cells of Meissner, as well as inner-core, outer-core and capsule of Pacinian corpuscles; and (c) dermal blood vessels, sweat glands and, occasionally, epidermis. The distribution of β/A4 IR matched that of βAPP, and no evidence of extracellular β/A4 IR was encountered. Present results demonstrate that PAPP, but not β/A4, is normally present in human glabrous (digital) skin. The potential clinical relevance of these findings is discussed.

Immunohistochemical localization of laminin and type IV collagen in human cutaneous sensory nerve formations

We used immunohistochemical techniques and monoclonal antibodies to localize two basement membrane components (laminin and type IV collagen) in the nerves and sensory nerve formations, or corpuscles, supplying human digital skin. Furthermore, neurofilament proteins, S-100 protein and epithelial membrane antigen were studied in parallel. In dermal nerve trunks, immunostaining for laminin and type IV collagen was found to be co-localized in the perineurium and the Schwann cells, the stronger immunoreactivity being at the external surface of the cells. In the Meissner digital corpuscles, the immunoreactivity for laminin and type IV collagen was mainly observed underlying the cell surface of lamellar cells, while the cytoplasm was weakly immunolabelled or unlabelled. Finally, within Pacinian corpuscles co-localization of the two basement membrane molecules was encountered in the inner core, intermediate layer, outer core and capsule. Laminin and type IV collagen immunoreactivities were also found in blood vessels and sweat glands, apparently labelling basement membrane structures. The present results provide evidence for the presence of basement membrane in all periaxonic cells forming human cutaneous sensory nerve formations, and suggest that all of them are able to synthesize and release some basement membrane components, such as laminin and type IV collagen. The possible role of laminin in sensory nerve formations is discussed.

Nerve growth factor receptor immunoreactivity in Meissner and Pacinian corpuscles of the human digital skin

The presence of nerve growth factor receptors (NGFr) in sensory nerve corpuscles of human digital skin, primarily Meissner and Pacinian corpuscles, was investigated immunohistochemically using two monoclonal antibodies directed against human-NGFr. To ensure the localization of NGFr immunoreactivity (IR) alternative sections to that processed for NGFr detection were assayed for neurofilament protein (NFP) and S-100 protein which selectively label the axon and the periaxonic specialized cells (lamellar cells of Meissner's corpuscles; inner-core cells of Pacinian corpuscles), respectively. Occurrence of NGFr IR was observed in both types of sensory corpuscles. In Meissner's corpuscles NGFr-IR was found in the lamellar cells, whereas in the Pacinian corpuscles the lamellae of the inner core, outer core, and capsule displayed NGFr IR. Moreover, a positive IR was observed in the central axon of some Pacinian corpuscles. However, remarkable differences were encountered among Pacinian corpuscles in the pattern of NGFr IR distribution. Present results demonstrate the presence of NGFr IR in sensory nerve corpuscles of the human digital skin, suggesting that NGFr could be involved in the concentration of NGF and in the conveying of this molecule from the cutaneous sources to the cell body of NGF-dependent primary sensory neurons. However, the mechanisms involved in this process remain to be clarified.

Endothelin-1 in Human Skin

The morphological distribution of endothelin-1 (ET-1) was investigated in human digital skin biopsies by immunohistochemistry using endothelin antisera, and by in vitro autoradiographic binding studies with 125I-labeled ET-1. In vitro studies on the responsiveness of cutaneous microvascular endothelium were carried out using human dermal microvascular endothelial cells (HDMEC) cultured from neonatal foreskin. Endothelin immunoreactivity was present in the endothelial cells of cutaneous blood vessels, in large and small arteries, veins in the dermis and capillaries, including those of the dermal papillae. Autoradiographic studies showed the presence of 125I-labeled ET-1 binding sites on the blood vessels and sweat glands. In confluent monolayers of HDMEC, ET-1 (100 pM-100 nM) significantly inhibited basal release of prostaglandin E2 (PGE2) (p less than 0.05), while causing a dose-dependent increase of intracellular cyclic adenosine monophosphate (cAMP) levels. cAMP accumulation, induced by 100 nM ET-1, was blocked by extracellular Ca2+ chelator EGTA, the intracellular Ca2+ chelator TMB-8, and dihydropyridine Ca(2+)-channel antagonist nifedipine (p less than 0.05), whereas ET-1 inhibition of PGE2 release was unaffected. These findings indicate that ET-1 may be important in the control of blood flow and vascular tone in the cutaneous microvasculature.

The early ontogeny of the afferent nerves and papillary ridges in human digital glabrous skin

The present study examines the early ontogeny of afferent nerves in human embryonic glabrous digital skin and documents the onset of cutaneous innervation and papillary (sweat duct) ridge formation by light and electron microscopy. The skin examined in this study was taken from 3 developmental stages of decreasing embryonic age: embryos older than 10 weeks estimated gestational age (EGA) representing the period of primary ridge formation, embryos of 8–9 weeks EGA representing the period immediately prior to ridge formation; and embryos 6–8 weeks EGA representing the period weeks before the onset of ridge formation. The earliest papillary ridges are present in 10 week EGA embryos, with small ridges present in two sites: the center of the proximal third and also at the tip of the distal phalangeal or apical pad. These papillary ridges typically contained Merkel cells. Papillary ridges formed progressively in a radial manner from these central foci. The proximal focus corresponds to the geometric center of the mature dermatoglyphic pattern of loops, arches, or whorls. This radial wave of ridge differentiation is discontinuous with the abrupt cessation of ridge formation responsible for the discontinuities in the mature papillary ridges and the corresponding dermatoglyphic print. Skin over the proximal and middle phalanges developed papillary ridges beginning in the 12th week. No papillary ridges could be identified in embryos of 8–9 weeks EGA, but a large number of growth cones are present in the superficial dermis subjacent to differentiating Merkel cells. The basal lamina of the epidermis was discontinuous wherever growth cones abutted Merkel cells. Merkel cells not directly associated with axons were also present in the epidermis of embryos of 8–9 weeks EGA. The embryos of 6–8 weeks EGA lack any sign of Merkel cells and/or melanocytes, but developing neurovascular bundles with axonal growth cones near the epidermis could be identified by light and electron microscopy. Presumptive Schwann and perineural cells are also seen in the dermis. We conclude that the developing afferent nerve fibers provide a grid which influences the temporal and/or spatial factors involved in the sequential onset and cessation of formation of papillary ridges. Thus the dermatoglyph can reflect the ontogeny of the afferent nervous system that occurred prior to papillary ridge development. These observations lend support to the concept that successive waves of afferent neural development have an important role in the spatial and temporal sequence of papillary ridge formation and thus the formation of both the dermatotopic map of the digits and the dermatoglyph.

Intraepidermal nerves in human digital skin.

Intraepidermal nerve fibers of human glabrous digital skin were investigated using a new silver impregnation method. Nerves were observed to enter the epidermis without regional preference, and to extend into the stratum granulosum. They are non-varicose (smooth) or varicose and range from less than 0.2 micron to approximately 2 micron in diameter, with varicosities up to 3 micron in diameter. Some axons branch profusely within the epithelium, giving off fine branches of differing diameters, while others appear to remain unbranched. At least some intraepidermal axons are fine branches of larger axons taking a horizontal course below the epithelial layer. Others are, at least topically, closely associated with Meissner's corpuscles. At 57 nerves per mm2 surface area, the density of intraepidermal nerves found in this investigation is much greater than that reported in recent publications, and agrees closely with values given in several older studies.

Immunohistochemical evidence of galanin in sensory nerves of human digital skin

Immunohistochemical evidence of galanin in sensory nerves of human digital skin

Localization of substance P-immunoreactive nerve fibers in the human digital skin.

Substance P-immunoreactive nerve endings were localized in human digital skin by the use of indirect immunohistochemical technique. It was found that substance P-like immunoreactivity was present in free nerve endings in the dermal papillae and in the epidermis. Some Meissner's corpuscles also contained substance P positive nerve endings. Furthermore, substance P-immunoreactive nerves were localized in close connection to sweat gland ducts and blood vessels. The functional significance of these findings was discussed with regard to pain mediation and inflammatory response.

Fine morphological characteristics and microtopography of the free nerve endings of the human digital skin.

Blocks of osmium-fixed human digital skin, representing a total area of 5 sq mm, were cut serially parallel to the skin surface starting with the epidermis for electron microscopical studies. At intervals, single 1-mu sections were taken and used for recording the microtopography of the receptor organs at various levels. Graphic reconstructions of 28 nerve endings were made. It was found that the digital skin accommodated up to 80 dermal papillae and three to four sweat duct orifices per square millimeter. Each papilla contained one to three fenestrated capillary loops. More than 60% of all papillae contained free nerve endings, sometimes up to five in a single papilla. As a result, up to 100 free nerve endings were counted per square millimeter. Intraepidermal endings were also present, one to five per square millimeter. No endings of any kind were found within the boundary zones between the papillary ridges and in the immediate vicinity of the sweat ducts. The origin of a free ending was marked by the perikaryon of a modified Schwann cell, which constituted the terminal cell of the Schwann sheath. The perikaryon was situated at the base of the dermal papilla, while its distal process provided the cytoplasmic sheath to the axon terminals and their branches. Sometimes two endings merged into a single receptor organ. The terminal Schwann cell frequently served as a rallying point of endings. Three morphological kinds of free endings were observed: open endings, beaded endings, and plain endings. The intraepidermal endings were the derivatives of the plain endings. The free endings in the digital skin had essentially vertical distribution. Each ending covered a minute surface area of skin in a punctate pattern. This is in contrast to the mode of distribution of the free (penicillate) endings of the human hairy skin. Each penicillus covers a large horizontal skin area and overlaps that of their neighbors.

Evidence against presence of chromaffin cells in human skin.

"Chromaffin cells" in human digital skin, described by previous workers, are readily recognized, but their granules do not produce the true chromaffin reaction of catecholamine, nor do they become green when subsequently stained with the Sevki technique. Although "chromaffin cells" differ morphologically from mast cells, they have similar tinctorial characteristics. Many cells were seen that could be accepted as either mast cells or "chromaffin cells." These cells in human skin are unaffected by intra-arterial reserpine and had the normal appearance in three patients with chronic degeneration of the sympathetic nerves. It is suggested that the "chromaffin cells" are mast cell variants and are not involv1ed in the autonomic effector mechanism.

THE DISTRIBUTION OF CHOLINESTERASE IN THE CUTANEOUS RECEPTOR ORGANS, ESPECIALLY TOUCH CORPUSCLES OF THE HUMAN FINGER

1. Cutaneous receptor organs of the human digital skin have been investigated for cholinesterase activity by a modification of the histochemical technique of Koelle and Friedenwald, and the findings have been analyzed in relation to the structure of the receptors. 2. It was found that in Meissner's corpuscles the nerves and their sheaths were negative, but the plasma membranes of the laminar cells and the intercellular substance gave strong positive reactions with either acetyl or butyryl substrates. 3. In Merkel's corpuscles, nerve endings and the associated Merkel's cells were negative, but the preterminal medullated nerve fibres gave positive reactions with the butyryl substrate. 4. In hairs, the terminal nerve fibres surrounding the epithelial sheaths gave positive reactions with both substrates. 5. Free nerve endings were negative but the cells related to the endings gave a positive reaction with both substrates. 6. Capillaries and sebaceous glands were negative, but adventitia of larger blood vessels gave positive reactions with the butyryl substrate. 7. The significance of the findings is discussed and a suggestion is made that both acetylcholine and its enzyme may constitute active agents in the synaptic transmission of a nervous impulse in Meissner's corpuscle.

The Anatomy and Histochemistry of the Arteriovenous Anastomosis in Human Digital Skin

The Anatomy and Histochemistry of the Arteriovenous Anastomosis in Human Digital Skin