Intraepithelial Axons Research Articles

The Effects of Diabetes and High-Fat Diet on Polymodal Nociceptor and Cold Thermoreceptor Nerve Terminal Endings in the Corneal Epithelium.

There is a substantial body of evidence indicating that corneal sensory innervation is affected by pathology in a range of diseases. However, there are no published studies that have directly assessed whether the nerve fiber density of the different subpopulations of corneal sensory neurons are differentially affected. The present study explored the possibility that the intraepithelial nerve fiber density of corneal polymodal nociceptors and cold thermoreceptors are differentially affected in mice fed with a high-fat high cholesterol (HFHC; 21% fat, 2% cholesterol) diet and in those that also have diabetes. The mice were fed the HFHC diet for the duration of the experiment (up to 40 weeks). Mice in the diabetes group had hyperglycaemia induced with streptozotocin after 15 weeks on the HFHC diet. Age-matched control animals were fed a standard diet. All corneal nerve fibers were labeled with a pan neuronal antibody (antiprotein gene product 9.5), and polymodal nociceptors and cold thermoreceptors were labeled with antibodies directed against transient receptor potential cation channel, subfamily V, member 1 and transient receptor potential cation channel subfamily M member 8, respectively. The mice fed a HFHC diet and those that in addition have hyperglycemia have similar reductions in corneal nerve fiber density consistent with small fiber neuropathy. Importantly, both treatments more markedly affected the intraepithelial axons of cold thermoreceptors than those of polymodal nociceptors. The results provide evidence that distinct subpopulations of corneal sensory neurons can be differentially affected by pathology.

Morphology of P2X3-immunoreactive nerve endings in the rat tracheal mucosa.

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3-immunoreactive nerve endings ramified into several branches that extended two-dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3-immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3-immunoreactive endings were beaded, rounded, or club-like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB-labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3-immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.

Anatomical Mapping and Density of Merkel Cells in Skin and Mucosae of the Dog

Merkel cells (MCs) are specialized cutaneous receptor cells involved with tactile sense. Although the distribution of MCs has been extensively studied in humans and rodents, their precise distribution and density throughout skin in the dog has not previously been determined. Knowledge of their distribution could facilitate understanding of their functions. By using of immunohistochemistry, density, and anatomical mapping of the MCs population in the dog skin was determined. Assessment of the MCs innervation was also achieved. Different patterns were noted in epidermis, hair follicles, or mucosa, including variable-sized clusters, linear or horse-shaped arrangements, and scattered and individualized cells. MCs revealed great variations in density and distribution over the body surface, with the highest numbers in oral mucosa and facial skin. There was no correlation of MCs density with age, sex, type of breed, coat type or pigmentation. Between 41 and 65% of MCs in hairy and glabrous skin and 8-18% of MCs in oral mucosa were in intimate contact with intraepithelial axon terminals. These findings indicate that canine MCs are numerous in sensory receptive areas and may be associated with the tactile sense in the dog. The present article enhances the knowledge of the skin structure in this species. Anat Rec, 299:1157-1164, 2016. © 2016 Wiley Periodicals, Inc.

Morphological Development and Expression of Neurotrophin Receptors in the Laryngeal Sensory Corpuscles

Morphological development of sensory structures in the laryngeal mucosa of postnatal rats was observed by use of immunohistochemistry for protein gene-product 9.5 (PGP9.5). Moreover, expression changes of high affinity neurotrophin receptors, TrkA, TrkB and TrkC, and low affinity neurotrophin receptor p75(NTR) were examined to elucidate the relationship to morphogenesis. Intraepithelial nerve endings and parent axons of the laminar endings with immunoreactivity for PGP9.5 have already appeared in the rat on embryonic day 18 (E18) as well as solitary chemoreceptor cells in the glottic cleft. According to neurotrophin receptors, TrkA immunoreactivity were observed on and after postnatal week 3 (3W) in the nervous sensory structures, that is, free nerve endings, laminar endings and sub- and intragemmal plexuses of the taste buds. In the laminar endings, TrkC immunoreactivity was also observed on and after 3W. According to the laryngeal sensory cells, the solitary chemoreceptor cells were immunoreactive to TrkA, TrkB, and TrkC on and after postnatal day 3 (P3). In the taste buds in arytenoid region, taste cells were immunoreactive for TrkA, TrkB, and TrkC on and after 3W, P14, and 3W, respectively. Immunoreactivity for p75(NTR) was observed on the surface of taste cells on and after P9. The results of the present study suggest that sensory structures in the laryngeal mucosa were developed on perinatal days to involve respiratory reflex, and that neurotrophin receptors may take part in the regulation and maintenance of sensory structures.

Morphological examination of the termination pattern of substance P-immunoreactive nerve fibers in human antral mucosa.

The termination pattern of substance P (SP)-containing axons in human antral mucosa was examined using immunohistochemical techniques at the light and electron microscopic level. SP-immunoreactive (IR) axons were found to extend towards the pit region of the glands, where intraepithelial axons were observed. Electron microscopy showed immunostained axon profiles in close contact with the basement membrane of surface mucous cells. Membrane-to-membrane contacts between labeled axons and myofibroblast-like cells were identified, and SP-IR axons that were apposed to the epithelium were also in contact with subjacent myofibroblast-like cells. The anatomical relationship between SP-IR axons and the cells of the muscularis mucosae was investigated by light microscopy. Immunoreactivity for alpha-smooth muscle actin (alpha-sma) was used to visualize the smooth muscle cells, and the alpha-sma-IR cells were found to create a network that surrounded the gastric glands. Immunostained varicose axons ran alongside and in close apposition to the labeled muscle strands. Ultrastructural examination showed close contacts between SP-IR axon profiles and smooth muscle-like cells. In conclusion, SP-containing neurons may be important for sensory and secretomotor functions in the human antral mucosa.

A Light and Electron Microscopic Anterograde WGA-HRP Tracing Study on the Sensory Innervation of Junctional and Sulcular Epithelium in the Rat Molar

The sensory innervation of junctional and oral sulcular epithelium was investigated by use of anterograde transport of wheat-germ agglutinin-horseradish peroxidase from the trigeminal ganglion. By light microscopy, labeled intra-epithelial nerve fibers were observed forming a dense plexus in the apical two-thirds of the junctional epithelium, with some fibers located near the enamel space. Occasional fibers extended coronally to the sulcus bottom. By electron microscopy, labeled intra-epithelial axon terminals or varicosities were demonstrated to be in close contact with both junctional epithelial cells and neutrophils. These varicosities, which were occasionally surrounded by the cytoplasmic processes of epithelial cells or neutrophils, frequently contained large granular and small clear vesicles. In contrast to the junctional epithelium, the oral sulcular epithelium was sparsely innervated, except for the transition region between the oral sulcular epithelium and the junctional epithelium, where a dense innervation by labeled intraepithelial fibers was found. These fibers extended as far as the stratum spinosum. Electron microscopy revealed mitochondria-filled profiles of varicosities between epithelial cells. This study shows differences in the distribution and ultrastructure of sensory nerves between the junctional and oral sulcular epithelia, and further provides morphological evidence that sensory nerves in the junctional epithelium come into contact not only with epithelial cells but also with neutrophils.

Calcitonin gene‐related peptide and corneal innervation: A developmental study in the rat

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.

Ultrastructure of the human vomeronasal organ

Virtually all vertebrates have a vomeronasal system whose involvement in pheromone detection plays a crucial role in reproduction. In humans, the vomeronasal organ has been assumed to be vestigial or absent and without functional significance. In the present study involving over 400 subjects, vomeronasal pits were observed in all individuals except those with pathological conditions affecting the septum. Electron microscopy of the adult human vomeronasal organ indicates the presence of two potential receptor elements in the pseudostratified epithelial lining: microvillar cells, and unmyelinated, intraepithelial axons. In addition, unmyelinated axons are common in the lamina propria surrounding the organ. They appear to constitute the components essential for a functional chemosensory system, and may thus provide the basis for a pheromone detection system as in other animals.

Peripheral patterns of calcitonin‐gene‐related peptide general somatic sensory innervation: Cutaneous and deep terminations

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.

Ultrastructural organisation of intraepithelial nerves in the human airway tract.

Intraepithelial nerves of human airway tract were studied by electron microscopy after conventional glutaraldehyde fixation. Specimens were obtained from five patients at three different airway levels--the trachea, the right upper lobe bronchus, and segmental bronchus. Intraepithelial axon profiles were located either near the basement membrane or close to the lumen but were rare in the intermediate area of the epithelium. Axon profiles close to the lumen were seen only in the central airways (levels 1 and 2), while profiles close to the basement membrane were seen in all three levels.

Effect of capsaicin on the intraperitoneal axons of the rat trachea

In vitro exposure of specimens of rat trachea to capsaicin at a concentration of 2 × 10 −4 g/ml was followed by a significant increase in the diameter and major changes in the vesicle content of intraepithelial axons with terminals containing a high proportion of large dense-cored vesicles and only scattered small vesicles, but had no effect on axons with terminals containing large numbers of closely-packed small vesicles. Twenty-four hours after in vivo treatment of rats with capsaicin at dose levels of 50 mg/kg, there was a highly significant reduction in the number of axons/unit length in the tracheal epithelium. This was attributed to similar patterns of degeneration of axons with terminals containing a high proportion of large dense-cored vesicles to those seen in the ureter and to the early elimination of the degenerating axons by phagocytosis by the epithelial cells.

Marking of olfactory axons of fishes by intravital staining with Procion brilliant yellow.

Olfactory axons of fishes can be traced after vital staining of the olfactory surface with the fluorescent dye Procion brilliant yellow M 4-RAN. Following selective accumulation by the receptor dendrites and perikarya the dye migrates intra-axonally over distances of more than 200 micrometer. Fluorescence microscope observation of formaldehyde fixed and paraffin sectioned preparations of the olfactory organ demonstrate clearly the system of intraepithelial axons and their first order bundles running toward the olfactory nerve.

Fine structure of sensory nerves in the rat cornea: An experimental nerve degeneration study

Fine structure of nerve fibres and terminals in the rat cornea was studied after fixation with glutaraldehyde and osmium tetroxide or with potassium permanganate. Superior cervical and/or ciliary ganglionectomy as well as ophthalmic neurotomy were performed in order to verify the origin of the corneal nerves. In the control corneas axon profiles with granular vesicles were observed in the stroma. Other axons containing agranular vesicles were found both in the stroma and in the epithelium. Superior cervical ganglionectomy abolished only the axons containing granular vesicles but did not affect the corneal blink reflex. Ciliary ganglionectomy induced more conspicuous changes in both stromal and intraepithelial axon profiles. The corneal reflex was, however, normal. Some of the axons containing agranular vesicles also showed degenerative changes. However, intact axon profiles with agranular vesicles were seen even after extirpation of both the ciliary ganglion and the superior cervical ganglion (combined autonomic ganglionectomy). On the other hand, all axon profiles with agranular vesicles disappeared after coagulation of the ophthalmic and maxillary nerves (sensory denervation). This operation destroyed most of the axons in all parts of the cornea and abolished the blink reflex. It is concluded that the axon profiles in the corneal epithelium, containing agranular vesicles, are sensory axons of the trigeminal nerve. A possible role of the vesicles in sensory nerves is discussed.

Structure and position of receptors within scales bordering the toes of gekkonids.

In gekkonids, the scales bordering the toes or the adjacent tissue possess suberpidermal and intraepithelial receptors in addition to setae-bearing organs. The position of subepidermal lamellated corpuscles seems to be correlated with the size of the species. The larger the adult animal the more frequently is this type of receptor found laterally in the toe. This can be explained in connection with the vibration-sensitive function of lamellated receptors. Intraepithelial axon terminals were found close to the setae-bearing sensilla in one species only. They are surrounded by numerous tonofibrils and may function as receptors for mechanical (pressure)-stimuli.

Ultrastructure of the corneal nerves in the rat.

The ultrastructure of the corneal nerves of the rat was studied in tissue fixed by immersion in and by perfusion with glutaraldehyde-containing fixatives. Of the four types of axonal terminal identified in the nerves, those with the features of adrenergic and cholinergic terminals were confined to the nerves at the limbus and were concentrated in the perivascular plexuses. The remaining two types of terminal were found on axons located in all parts of the cornea and on both intraepithelial axons located in all parts of the cornea and on both intraepithelial axons and axons in the stromal nerves. Of these, one contained the numerous mitochondria which occur in the terminals of axons associated with known mechanoreceptors and the second contained variable and often small numbers of both clear and large dense-cored vesicles. While most of the mitochondria-containing terminals were seen in nerves located near the periphery,vesicle-containing terminals were numerous in all of the nerves,and especially in those in the avascular cornea. In material fixed by immersion in glutaraldehyde-paraformaldehyde,the vesicle-containing terminals appeared to be dilated,but in material fixed by perfusion there was little evidence of any increase in the diameter of the axons in the terminal regions. The structure of the terminals was compared with that of the terminals of axons identified in the nerves of the skin and the urinary tract and the differences in the vesicle content of the terminals to those reported in other studies of the corneal nerves was related to the use of different fixation procedures. The possibility that axons possessing such terminals are identical with the beaded axons and both the cholinesterase-positive and fluorescent axons demonstrated in light microscopical studies of the corneal nerves is discussed, and the widespread distribution of the axons in the cornea is equated with the hypothesis that they are afferent in nature and represent the peripheral receptors for pain impulses.

Granular "endocrine" cells in avian respiratory epithelia.

An electron microscopic investigation of the extrapulmonary respiratory tract of embryos and chick of the domestic fowl (Gallus domesticus) has demonstrated for the first time in birds the presence here of a small number of epithelial cells characterised by an aminecontaining type of granule. These granular cells were scattered singly throughout the trachea, syrinx and primary bronchi and seemed more numerous in the caudal part of the airway. In favourable planes of section a small part of the cell was in contact with the luminal surface of the epithelium. The characteristic granular vesicles (approximate diameter 140 nm) appeared to be randomly distributed in the cytoplasm and there was no concentration of vesicles close to the plasma membrane. One of the cells was closely associated with an intraepithelial axon. By fluorescence microscopy, a small number of cells with a similar shape and distribution to the granular cells was observed after administration of 3,4-dihydroxyphenylalanine which may indicate the presence of an amine handlign mechanism in these cells. It is suggested that the granular cells belong to the APUD system of endocrine cells and that they may be modulated by the concentration of gas in the airways.

Intraepithelial axons in the avian trachea.

The innervation of the epithelium of the avian trachea was investigated in the domestic fowl (Gallus domesticus) with cholinesterase and fluorescence techniques and electron microscopy. In the subepithelial tissues there were many nerves associated with acetylcholinesterase but only a very few containing catecholamines. The nerves were arranged in two interconnected plexuses, one plexus of myelinated and unmyelinated axons lying external to the tracheal cartilages, and another plexus of unmyelinated axons and ganglion cells lying between the cartilages and the epithelium. The presence of intraepithelial axons was established with the electron microscope. The majority of the intraepithelial axons lay near to the basal cells and the ciliated cells and some approached close to the surface of the epithelium. Two types of vesicle, agranular and granular occurred in the axon profiles. Convincing evidence for synapses between intraepithelial axons and epithelial cells was not seen. A sensory function is suggested for these axons.