Nerves and nerve endings in the skin of tropical cattle.

The nerves and nerve endings in the skin of tropical cattle were studied using histological and histochemical techniques. Many nerve trunks and fibres were present in the reticular and papillary dermis in both hairy and non-hairy skin sites. In non-hairy skin locations such as the muzzle and lower lip, encapsulated endings akin to Krause and Ruffini end bulbs, which arise from myelinated nerve trunks situated lower down the dermis were observed at the upper papillary layer level. Some fibre trunks seen at this level extended upwards to terminate within dermal papillae as bulb-shaped longitudinally lamellated Pacinian-type endings, while other onion-shaped lamellated nerve structures were located either within dermal papillae or near the dermo-epidermal area. Intraepidermal free-ending nerve fibres, appearing non-myelinated were observed in areas with thick epidermis. Intraepidermal free-ending nerve fibres, appearing non-myelinated were observed in areas with thick epidermis. On hairy skin sites, however, organized nerve endings or intraepidermal nerve endings were not readily identifiable.     

Time Course and Characteristics of the Capacity of Sensory Nerves to Reinnervate Skin Territories outside Their Normal Innervation Zones

Factors involved in the outcome of regeneration of the saphenous nerve after a cut or crush lesion were studied in adult rats with electrophysiological recordings of low-threshold mechanoreceptor activity and plasma extravasation of Evans blue after electrical nerve stimulation that activated C fibers.

In the first series of experiments, saphenous and sciatic nerve section was combined with anastomosis of the transected proximal end of the saphenous nerve to the distal end of the cut tibial nerve. Regeneration of saphenous nerve fibers involved in plasma extravasation and low-threshold mechanoreceptor activity in the glabrous skin was observed 13 weeks after nerve anastomosis. Substance P-, calcitonin gene-related peptide-, and protein gene product 9.5 (PGP-9.5)-immunoreactive (IR) thin epidermal and dermal nerve endings, as well as coarse dermal PGP-9.5-IR nerve fibers and Meissner corpuscles and Merkel cell-neurite-like complexes, were observed in the reinnervated glabrous skin at this time.

In a second series of experiments, the time course of the regeneration of saphenous nerve axons to the permanently sciatic-nerve-denervated foot sole was examined. Saphenous-nerve-induced plasma extravasation and low-threshold mechanoreceptor activity in the saphenous nerve were found in the normal saphenous nerve territory 2, 3, 4, and 6 weeks after sciatic nerve cut combined with saphenous nerve crush in the left hindlimb. Saphenous-nerve-induced plasma extravasation was also present in the glabrous skin normally innervated by the sciatic nerve 3, 4, and 6 weeks after the sciatic cut/saphenous crush lesion. However, no low-threshold mechanoreceptor activity was detected in the saphenous nerve when the glabrous skin area was stimulated.

In a third series of experiments, the fate of the expansion of the saphenous nerve territory after saphenous nerve crush was examined when the crushed sciatic nerve had been allowed to regenerate. Nerve fibers involved in plasma extravasation were observed in the glabrous skin of the hindpaw after saphenous nerve, as well as after tibial nerve, C-fiber stimulation 3, 12, and 43 weeks after the saphenous crush/sciatic crush lesion.

Low-threshold mechanoreceptors from the regenerated saphenous nerve, which primarily innervates hairy skin, seem to be functional in the glabrous skin if the axons are guided by the transected tibial nerve by anastomosis. Furthermore, the results indicate that fibers from the regenerating saphenous nerve that have extended into denervated glabrous skin areas can exist even if sciatic nerve axons are allowed to grow back to their original territory.     

Intrinsic innervation of the rat knee joint articular capsule and ligaments

In spite of the practical importance of having a detailed knowledge of knee joint innervation to understand the pathophysiologic aspects, little information is now available concerning the density and pattern of the nerve fibres which are distributed to it. The present study has been designed to investigate the density and distribution of nerve fibres and receptor corpuscles in the knee joint articular capsule, cruciate and collateral ligaments in the rat, using the acetylcholinesterase (AChE) histochemical in toto staining technique. The investigation was performed on male Wistar rats of 3 months of age, some of which had been treated with capsaicin to deplete their afferent 'C' fibres of their content of neuropeptides. AChE-positive nerve fibres and different types of receptor corpuscle endings were found within articular capsule and ligaments. The highest density of AChE-positive nerve fibres was noticeable in the fibular collateral ligament followed by the tibial collateral ligament, the posterior cruciate ligament, the anterior cruciate ligament and the articular capsule. In the articular capsule the number of type I endings was higher than in the ligaments. The opposite is true for the other type of receptor corpuscles found as well as for nerve endings. Capsaicin treatment significantly reduced the density of AChE-positive nerve fibres in knee joint ligaments but did not affect nerve fibres in the articular capsule. Moreover, it caused the disappearance of some kind of receptor corpuscles within the collateral and cruciate ligaments. The above data collectively suggest that the AChE in toto staining technique may represent a good method for investigating joint innervation and that a significant percentage of nerve fibres supplying knee joint ligaments is represented by C fibre afferents.     

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

Summary In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.     

Light-evoked somatosensory perception of transgenic rats that express channelrhodopsin-2 in dorsal root ganglion cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain.     

Structure of the sensory innervation of the anal canal in the pig. A light- and electron-microscopical study

The sensory innervation of the anal canal of the pig was investigated by light and electron microscopy. The distribution of the different types of sensory nerve endings correlates with the histology of different zones: (1) After the rectal mucosa there was a zone lined with nonkeratinized stratified squamous epithelium. (2) A middle zone was lined with keratinized stratified squamous epithelium. Here the dermis already showed a papillary and reticular layer. (3) The last zone showed hairy skin with a high hair density. The following nerve endings were found: Free nerve endings reached the stratum superficiale in nonkeratinized squamous epithelium and the stratum granulosum in the keratinized squamous epithelium. Dermal free nerve endings were found in all zones near the epithelium and two different types were identified as those derived from C-fibers and those from A-delta-fibers. Merkel nerve endings showed different features depending on their location. Few Merkel-like cells were found in the epithelium of the anal crypts. Typical Merkel Tastscheiben were located at the base of epithelial ridges or pegs in zones 2 and 3. The number of Merkel cells varied up to 200. The myelinated afferent fiber supplied 10-15 Merkel cells. Merkel cells were also found regularly in the outermost layer of the external rooth sheath of hair follicles at about the same level as perifollicular nerve endings. Lamellated corpuscles were found in the dermis of all zones except the cranial part of zone 1, where the anal crypts are located. Generally they consisted of a central nerve terminal which may be branched. Each terminal was surrounded by an inner core of concentrically arranged lamellae of the terminal Schwann cell and one or several inner cores were included in a capsule of perineural cells. The size of the corpuscle, the regularity of the inner core and the number of capsular layers depended on the location of the corpuscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new combination of methods for the localization,identification, and three-dimensional reconstruction of the sensory endings of articular afferents characterized by electrophysiology

A combination of methods is described to identify and reconstruct corpuscular and non-corpuscular sensory endings of group II and group III nerve fibers following functional examination by electrophysiology. Afferent units activated by electrical stimulation of the medial articular nerve of the cat's knee were analyzed by single fiber recordings and characterized by their responsiveness to mechanical stimuli. The receptive fields of the units were closely demarcated by fine needles when the responses elicited by insertion of the needles were being recorded. After fixation, the tissue around the demarcated field was dissected and histologically processed. Series of semithin sections were cut from the embedded tissue blocks containing the receptive fields. Corpuscular endings of group II fibers and peripheral myelinated group III nerve fibers, presumably corresponding to the characterized units, were identified by light microscopy of semithin sections and localized within the demarcated area. Non-corpuscular endings were identified by electron microscopy of ultrathin sections cut in alternation with, or after re-embedding of, semithin sections. Morphometric analysis of ultrathin section series allowed the measurement of parameters such as the mean axon diameter and the organelle content of the sensory endings. The methods described are appropriate for collecting data that correlate the structural and functional characteristics of sensory endings in deep tissues.     

Spatiotemporal dynamics of re-innervation and hyperinnervation patterns by uninjured CGRP fibers in the rat foot sole epidermis after nerve injury

ABSTRACT: The epidermis is innervated by fine nerve endings that are important in mediating nociceptive stimuli. However, their precise role in neuropathic pain is still controversial. Here, we have studied the role of epidermal peptidergic nociceptive fibers that are located adjacent to injured fibers in a rat model of neuropathic pain. Using the Spared Nerve Injury (SNI) model, which involves complete transections of the tibial and common peroneal nerve while sparing the sural and saphenous branches, mechanical hypersensitivity was induced of the uninjured lateral (sural) and medial (saphenous) area of the foot sole. At different time points, a complete foot sole biopsy was taken from the injured paw and processed for Calcitonin Gene-Related Peptide (CGRP) immunohistochemistry. Subsequently, a novel 2D-reconstruction model depicting the density of CGRP fibers was made to evaluate the course of denervation and re-innervation by uninjured CGRP fibers. The results show an increased density of uninjured CGRP-IR epidermal fibers on the lateral and medial side after a SNI procedure at 5 and 10 weeks. Furthermore, although in control animals the density of epidermal CGRP-IR fibers in the footpads was lower compared to the surrounding skin of the foot, 10 weeks after the SNI procedure, the initially denervated footpads displayed a hyper-innervation. These data support the idea that uninjured fibers may play a considerable role in development and maintenance of neuropathic pain and that it is important to take larger biopsies to test the relationship between innervation of injured and uninjured nerve areas.     

Sensory cells in the head skin of pond snails

Summary Several types of receptor endings were identified with scanning electron microscopy and silver-impregnation techniques in the skin of the tentacles, lips, dorsal surface of the head and mouth region of the pond snails Lymnaea stagnalis and Vivipara viviparus. Sensory endings at the tips of dendrites of primary receptor neurones, scattered below the epithelium, differ in structure, i.e., the endings exposed to the surface of the skin possess different proportions of cilia and microvilli, which vary in number, length, and packing. Type-I endings have microvilli and a few (1–5) cilia, 5–12 m in length. Type-2 endings have abundant (20–40), interwoven long (9–12 m) cilia and random microvilli. Type-3 endings show typical packing of 10–25 cilia in the form of bundles or brushes. They may be composed either of long (9–18 m) or short (2–7 m) cilia, or of both long and short ones. Microvilli here are absent. Type-4 endings have only microvilli. Two other types of skin receptors do not extend their sensory endings to the surface and can be indentified only in silver-stained preparations. Type-5 endings are branching dendrites of skin receptors cells that terminate among epithelial cells. In type-6, the sensory endings also terminate among epithelial cells but their cell bodies are located outside of the skin. In both species all skin regions examined possess the receptors of all six types differing only in their relative proportion. Possible functional roles of different receptors are discussed.     

Morphofunctional restoration of a nerve trunk by filling the defect with a blood vessel

Dynamics of morphologic and functional restoration of the nerve trunk and separate receptive skin formations have been investigated in white rats after a sutureless connection of the cut nerve by means of the implanted artery. For testing the reparative processes neurohistological and electrophysiological techniques have been used. In spite of a relatively early restoration of the integrity of the injured nerve and reaching the nervous fibers up to the skin, functional organization of the dermal sensory apparatus is observed only in a year after the operation.     

Skin mechanoreceptor functioning in the white rat during transcranial electrostimulation

Functional properties of skin afferents were studied by the method of registration of spike activity from single nerve fibres, innervated non-hairy skin in white rats. It was found that mechanoreceptor units varied by the threshold intensity of applied stimulus. These units were divided to three functional groups: low-, medium- and high-threshold units. Transcranial electrical stimulation (TES) strongly affected on the functional characteristics of skin afferents. Full suppression of responses was observed in 18-20 min. irrespective of the category of units. A local intracutaneous injection of naloxon eliminated the effect of TES. It is supposed that endogenous opioid peptides regulate an afferent discharge on the level of sensory endings during TES.     

A rapid and dynamic regulation of GDNF-family ligands and receptors correlate with the developmental dependency of cutaneous sensory innervation.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are members of the transforming growth factor-beta family and have been shown to elicit neurotrophic effects upon several classes of neurons including dopaminergic neurons, motoneurons, parasympathetic, sympathetic as well as primary sensory neurons. However, there is little information available on their roles in cutaneous innervation. Herein, we have studied the regulation of gdnf, ntn and the GDNF family receptors and examined their role in the development of facial cutaneous innervation in GDNF mutant mice. A dynamic spatial and temporal regulation of gdnf, ntn and their ligand binding receptors within the follicle-sinus complex correlate with development of distinct subclasses of sensory nerve endings. Furthermore, development of NGF-dependent myelinated mechanoreceptors, i.e. reticular and transverse lanceolate endings also require GDNF during ending formation and maintenance. In addition, ligand and receptor association seems to be intricately linked to a local Schwann cell-axon interaction essential for sensory terminal formation. Our results suggests that functionally specified nerve endings depend on different GDNF family members and that in contrast to neurotrophins, this family of neurotrophic factors may be acting at local sites of terminal Schwann cell-axon growth cone interactions and that they collaborate with neurotrophins by supporting the same populations of neurons but at different times in development.     

The role of semaphorin3A in myogenic regeneration and the formation of functional neuromuscular junctions on new fibres

Current research on skeletal muscle injury and regeneration highlights the crucial role of nerve–muscle interaction in the restoration of innervation during that process. Activities of muscle satellite or stem cells, recognized as the ‘currency’ of myogenic repair, have a pivotal role in these events, as shown by ongoing research. More recent investigation of myogenic signalling events reveals intriguing roles for semaphorin3A (Sema3A), secreted by activated satellite cells, in the muscle environment during development and regeneration. For example, Sema3A makes important contributions to regulating the formation of blood vessels, balancing bone formation and bone remodelling, and inflammation, and was recently implicated in the establishment of fibre‐type distribution through effects on myosin heavy chain gene expression. This review highlights the active or potential contributions of satellite‐cell‐derived Sema3A to regulation of the processes of motor neurite ingrowth into a regenerating muscle bed. Successful restoration of functional innervation during muscle repair is essential; this review emphasizes the integrative role of satellite‐cell biology in the progressive coordination of adaptive cellular and tissue responses during the injury‐repair process in voluntary muscle.     

The roles of receptor tyrosine kinases and their ligands in the wound repair process

Cutaneous wound repair is a tightly regulated and dynamic process involving blood clotting, inflammation, formation of new tissue, and tissue remodeling. Gene expression profiling of mouse and human wounds as well as first proteomics studies have identified a large number of genes and proteins that are up- or downregulated by skin injury, and some of them have been functionally characterized in animal models of wound repair. Among the key regulators of wound repair are growth factors, which control migration, proliferation, differentiation and survival of cells at different stages of the healing process. This review summarizes the results of functional studies performed in mammals that have identified important roles of receptor tyrosine kinases and their ligands in wound repair.     

Effects of ultraviolet radiation on human cutaneous nerve fibres

Due to an increasing number of skin diseases as a result of exposure to ultraviolet (UV) radiation, it is necessary to evaluate the effectiveness of new skin care formulations with broad-spectrum sunscreens.
Objectives:  This study aims to assess the status of nerve fibres in healthy human skin, to quantify effects of UV radiation on nerve endings, and to evaluate neuroprotective effects of new skin care formulations against UV exposure damage.
Methods:  Samples were obtained from 34 female patients enrolled for plastic surgery and were immediately treated (10 min) with three emulsions: Cream 1, Cream 2 (placebo) and a sunscreen with sun protection factor 15 (SPF15). Control samples and those treated with the cream emulsions were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody (anti-human CD56/NCAM). Cell damage was assessed by image analysis.
Results:  Several cellular nervous structures were identified in the skin samples, including free nerve endings. UVA and UVB significantly decreased (40–60%) density of nerve endings in the control samples and those treated with placebo (Cream 2) or SPF15 (all P  < 0.001). Cream 1 completely blocked effects of UV radiation on nerve endings ( P  > 0.05 vs. control).
Conclusions:  Quantification of cell damage induced by UV radiation provides useful information for identification of new skin care compounds with neuroprotective properties.     

Melanocytes: morphological basis for an exteroceptive sensory system for monitoring ultraviolet radiation

From the conventional medical perspective, melanocytes have been traditionally viewed as epidermal responders/reactors to ultraviolet radiation. In this paper we have begun an analysis of the functional significance of melanocytes as monitors for ultraviolet radiation with their neuronal, endocrinological and immunological intercalations. This preliminary study was performed using as a human model unilateral sural nerve sensory blockade before and after ultraviolet exposure to both feet while the unblocked foot acted as the control. Full thickness surgical skin biopsies were taken of (I), normal pre-exposure skin, in the center of the two centimeter exposure area of both the sural nerve blocked area (II-III) and control (IV-V) one centimeter outside the exposed area in both the sural nerve blocked foot and the control or non-blocked foot. The objective clinical, blinded morphological and immuno-histochemical data from this experiment support the initial conclusion that neuronal connection is necessary for the normal ultraviolet exposure dermal reaction. Based upon this study, we further propose the existence of an exteroceptive sensory system in which melanocytes, with direct nervous system connection initiate an ultraviolet radiation reactive response that mobilizes both conventional endocrine and immune pathways.     

Neuromuscular rehabilitation by treadmill running or electrical stimulation after peripheral nerve injury and repair.

Numerous studies have been devoted to the regeneration of the motor pathway toward a denervated muscle after nerve injury. However, the regeneration of sensory muscle endings after repair by self-anastomosis are little studied. In previous electrophysiological studies, our laboratory showed that the functional characteristics of tibialis anterior muscle afferents are differentially affected after injury and repair of the peroneal nerve with and without chronic electrostimulation. The present study focuses on the axonal regeneration of mechano- (fibers I and II) and metabosensitive (fibers III and IV) muscle afferents by evaluating the recovery of their response to different test agents after nerve injury and repair by self-anastomosis during 10 wk of treadmill running (LSR). Data were compared with control animals (C), animals with nerve lesion and suture (LS), and animals with lesion, suture, and chronic muscle rehabilitation by electrostimulation (LSE) with a biphasic current modulated in pulse duration and frequency, eliciting a pattern mimicking the activity delivered by the nerve to the muscle. Compared with the C group, results indicated that 1) muscle weight was smaller in LS and LSR groups, 2) the fatigue index was greater in the LS group and smaller in the LSE group, 3) metabosensibility remained altered in the LS and LSE groups, and 4) mechanosensitivity presented a large increase of the activation pattern in the LS and LSE groups. Our data indicated that chronic muscle electrostimulation partially favors the recovery of muscle properties (i.e., muscle weight and twitch response were close to the C group) and that rehabilitation by treadmill running also efficiently induced a better functional muscle afferent recovery (i.e., the discharge pattern was similar to the C group). The effectiveness of the chronic electromyostimulation and the treadmill exercise on afferent recovery is discussed with regard to parameters listed above.     

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.     

Electron microscopic studies of the pressoreceptor fields of the carotid sinus of the dog

Summary In the dog, pressosensitive endings of the sinus nerve extend along the border between the adventitia and media of the carotid sinus wall. The axon endings, containing a great number of mitochondria, can be divided into small (600–2,000 nm) and large (6,000–8,000 nm) end swellings. In the terminal region the pressosensitive fibers are surrounded by ramified and highly structured Schwann terminal cells. The topographic location in relation to elastic and collagenous tissue indicates a functional relation between vascular wall and the activity of receptor tissue. A functional connection between receptors and efferent nerve endings in the immediate surroundings has been discussed in this report. Several axon endings contain variable amounts of glycogen which is regarded as an indication for the inactive metabolic state of the ending. Axonal swellings demonstrate considerable modification in structure, such as loss of structural integrity in mitochondria, the formation of lamellar fields, vesicular irregularities and disintegration of axoplasm, all of which are considered as the morphological expression of wearing out, degeneration and possibly regeneration.This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 104)Dedicated to Professor W. Bargmann on the occasion of his 70th birthday