Types Of Nerve Fibers Research Articles

Re-evaluation and quantification of the different sources of nerve fibres supplying the rat eye.

The denervation and/or the removal of peripheral nerve ganglia are useful surgical techniques for studying the source and distribution of peripheral nerves in all organs, including the eye. The amount and distribution of the remaining nerve fibres supplying the eye (after sectioning of various types of nervous fibres and/or removal of nerve ganglia) were evaluated in the rat. Male Sprague-Dawley rats were anaesthetized and one or more of the following nervous tissues were removed: superior cervical ganglion, main ciliary ganglion, pterygopalatine ganglion, trigeminal ganglion and the ophthalmic-maxillary nerve. In some animals, chemical sympathectomy was performed by administration of 6-OH dopamine. The eyes were cut in serial sections, but only three regions (cornea, iris and choroid) were harvested and submitted for various nerve fibre staining techniques. The results were quantified and statistically analysed. Superior cervical ganglionectomy and/or chemical sympathectomy induced the destruction of almost all the catecholaminergic nerve fibres in the three examined regions of the rat eye. Removal of the ciliary ganglion (partial parasympathectomy) caused the destruction of about 60% of the cholinergic nerve fibres of the same regions of the rat eye, while subtotal parasympathectomy destroyed about 80% of the cholinergic nerve fibres. Surgical transsection of the ophthalmo-maxillary nerve or the removal of the trigeminal ganglion led to a degeneration of almost all sensitive nerve fibres of the three examined regions of the rat eye. The denervation experiments confirmed the presence of the different types of nerve fibres (sympathetic, parasympathetic and sensitive) in the three studied structures of the rat eye.

Modern aspects of cutaneous neurogenic inflammation.

Recent findings have shed new light on the role of peripheral nerves in the skin and established a modern concept of cutaneous neurobiology. Closely related monodirectional and/or bidirectional pathways exist in which the central and peripheral nervous system, the endocrine and immune system, and almost all skin cells are involved. Information is emerging about the factors involved in these immunomodulatory mechanisms, which are defined as neuropeptides, neurotransmitters, neurotrophins, and neurohormones. The interaction between peripheral nerves and the immune system is mediated by different types of cutaneous nerve fibers that release neuromediators and activate specific receptors on target cells in the skin such as keratinocytes, mast cells, Langerhans cells, microvascular endothelial cells, fibroblasts, and infiltrating immune cells. These interactions influence a variety of physiologic and pathophysiologic functions including cellular development, growth, differentiation, immunity, vasoregulation, leukocyte recruitment, pruritus, and wound healing. A variety of mechanisms lead to the termination of cellular responses to released neuropeptides under physiologic circumstances. Herein, we highlight some of the recent advances of neurocutaneous biology and discuss the role of nerves in mediating cutaneous inflammation. Understanding the mechanisms and the factors controlling neuromediators and their receptors and degrading enzymes will lead to the identification of novel therapeutic targets for the treatment of cutaneous diseases.

Fetal lateral ganglionic eminence attracts one of two morphologically different types of tyrosine hydroxylase-positive nerve fibers formed by cultured ventral mesencephalon.

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3-21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 microm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.

Age-Related Changes in Catecholaminergic Nerve Fibers of Rat Heart and Coronary Vessels

Background: Age-related changes of catecholaminergic nerve fibers supplying the myocardium and the coronary vessels were studied in adult and in old rats. Objective: The purpose of this study was to investigate whether there are quantitative changes in myocardial and/or coronary catecholaminergic nerve fibers and related neurotransmitters. Methods: Samples of myocardium and/or coronary vessels were studied by means of glyoxylic acid induced fluorescence, aldehyde-induced fluorescence, and neuropeptide Y immunoreactivity. All morphological results were submitted to quantitative analysis of images. Biochemical dosage of proteins and of norepinephrine was also performed, and morphological data were compared with biochemical ones. Finally, all data were subjected to statistical analysis. Results: Our findings are indicative of an age-related decrease of all types of fluorescent sympathetic nerve fibers in rat heart and coronary arteries. Moreover, our data show that aging induces a strong decrease of norepinephrine levels in tissue homogenates of rat heart and coronary vessels. The majority of the catecholaminergic nerve fibers that show strong age-related changes are not contiguous with contractile cells of myocardium and/or of vascular wall. In fact, these fibers are located in adventitial (or epicardial) and in endothelial (or endocardial) layers. Conclusion: The decrease of the catecholaminergic innervation of heart and coronary vessels of old rats can be in close relationships to the poor cardiac and/or coronary functions observed in old animals.

Lateral line reception in still- and running water.

The lateral line of fish is composed of neuromasts used to detect water motions. Neuromasts occur as superficial neuromasts on the skin and as canal neuromasts in subepidermal canals. Fibres of the lateral line nerves innervate both. There have been extensive studies on the responses of lateral line nerve fibres to dipole stimuli applied in still water. However, despite the fact that many fish live in rivers and/or swim constantly, responses of lateral line nerve fibres to dipole stimuli presented in running water have never been recorded. We investigated how the peripheral lateral line of still water fish ( Carassius auratus) and riverine fish ( Oncorhynchus mykiss) responds to minute sinusoidal water motions while exposed to unidirectional water flow. Both goldfish and trout have two types of posterior lateral line nerve fibres: Type I fibres, which most likely innervate superficial neuromasts, were stimulated by running water (10 cm s(-1)). The responses of type I fibres to water motions generated by a vibrating sphere were masked if the fish was exposed to running water. Type II fibres, which most likely innervate canal neuromasts, were not stimulated by running water. Consequently, responses of type II fibres to a vibrating sphere were not masked under flow conditions.

Presence and distribution of sensory nerve fibers in human peritoneal adhesions.

To assess the distribution and type of nerve fibers present in human peritoneal adhesions and to relate data on location and size of nerves with estimated age and with clinical parameters such as reports of chronic pelvic pain. Peritoneal adhesions are implicated in the cause of chronic abdominopelvic pain, and many patients are relieved of their symptoms after adhesiolysis. Adhesions are thought to cause pain indirectly by restricting organ motion, thus stretching and pulling smooth muscle of adjacent viscera or the abdominal wall. However, in mapping studies using microlaparoscopic techniques, 80% of patients with pelvic adhesions reported tenderness when these structures were probed, an observation suggesting that adhesions themselves are capable of generating pain stimuli. Human peritoneal adhesions were collected from 25 patients undergoing laparotomy, 20 of whom reported chronic pelvic pain. Tissue samples were prepared for histologic, immunohistochemical, and ultrastructural analysis. Nerve fibers were characterized using antibodies against several neuronal markers, including those expressed by sensory nerve fibers. In addition, the distribution of nerve fibers, their orientation, and their association with blood vessels were investigated by acetylcholinesterase histochemistry and dual immunolocalization. Nerve fibers, identified histologically, ultrastructurally, and immunohistochemically, were present in all the peritoneal adhesions examined. The location of the adhesion, its size, and its estimated age did not influence the type of nerve fibers found. Further, fibers expressing the sensory neuronal markers calcitonin gene-related protein and substance P were present in all adhesions irrespective of reports of chronic abdominopelvic pain. The nerves comprised both myelinated and nonmyelinated axons and were often, but not invariably, associated with blood vessels. This study provides the first direct evidence for the presence of sensory nerve fibers in human peritoneal adhesions, suggesting that these structures may be capable of conducting pain after appropriate stimulation.

Central responses evoked by tactile stimulation: technical note

The electrical stimulation of peripheral nerves is one of the commonest ways to study the sensory pathways from the peripheral nerve to the cortex. Such stimulation, however, involves all types of nerve fibres regardless of their specific function. In the past, we devised a technical procedure to record peripheral responses by selectively activating fibres innervating cutaneous mechanoreceptors. In the present paper, we illustrate the method that we developed to record cortical responses evoked by mechanical stimulation. The analysis of the tactile evoked potentials needs an adequate procedure to acquire and to elaborate the signals. Difficulties in setting these parameters make mechanical SEPs not routinely performed. This study is a first step in the development of a reliable system to record mechanical SEPs for clinical purposes.

Composition of the medial and posterior articular nerves of the mouse knee joint

The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 - 28 nerve fibers consisting of 63 - 24 unmyelinated and 12 - 6 myelinated fibers. The PAN was composed of 195 - 50 nerve fibers, namely 129 - 28 unmyelinated and 66 - 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 w m with a maximum between 0.3 and 0.6 w m. The myelinated nerve fibers in the MAN ranged from 1 to 8 w m with a peak between 2 and 5 w m. In the PAN, their diameters ranged from 1 to 12 w m with a clearly visible peak at 4-5 w m and a plateau at 8-9 w m that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.

Distribution of peptidergic nerve fibres in the guinea pig trabecular meshwork.

A quantitative analysis of peptidergic nerve fibres located in the trabecular meshwork of the guinea pig has been performed. Our results confirm that this structure contains VIP-, NPY- and substance P-like immunoreactivity as major neurotransmitters. These findings were obtained using immunohistochemical techniques. For this purpose serial sections of the eye were stained by immunohistochemistry for each of three neurotransmitters and stained sections were analysed by quantitative image analysis. Our findings demonstrate that SP-positive, NPY-positive and VIP-positive nerve fibres occupy 11.2, 4.9 and 2.4%, respectively, of the observed area (expressed as conventional units, C.U.) in the trabecular meshwork of the guinea pig eye. It is relevant to emphasize that the area containing these three types of peptidergic nerve fibres appears to be large (18.5 +/- 6.6 C.U.) in proportion to the total observed area. The innervation of the drainage angle of the guinea pig eye has been well described by many authors. This is the first study to report quantitative measurements of three types of peptidergic nerve fibres identified and measured in this area. The presence of these three neurotransmitters in the trabecular meshwork of guinea pig eye suggests their possible participation in aqueous humor regulation.

Prevalence and ultrastructural morphology of axosomatic synapses on spiral ganglion cells in humans of different ages

Axosomatic synapses were found on human spiral ganglion cells (HSGCs). Ultrastructural characterization and calculation of the prevalence of these synapses were performed by electron microscopic semi-serial sections of both type I and type II HSGCs, in specimens from subjects of ages 1 day, 14 days, 21 years and 51 years. Synapses on type I HSGCs were extremely rare. In contrast, axosomatic synapses were present on approximately 50% of type II HSGCs of a young adult. This prevalence seemed to vary by age. Thus, no synapses were found in a 1-day old neonate, few in a 14-day old, and on approximately 15% of the type II SGCs from a 51-year old specimen. The origin of the nerve fibers synapsing on the type II HSGCs could not be determined. In view of the fact that some of the fibers projected from the intraganglionic spiral bundle, which is known to contain olivocochlear efferents, these fibers may represent an efferent pathway to the spiral ganglion. However, since there was morphological evidence of more than one type of nerve fiber synapsing on type II HSGCs, other neural origins must be considered. Although the physiological function of these synapses is unknown, they may mediate pre-synaptic neural modulation of the type II HSGCs at the level of the spiral ganglion.

Propioceptores articulares y musculares

La función de los mecanorreceptores de las articulaciones y músculos se considera asociada a
 la propiocepción. Sin embargo, existen evidencias de que la propiocepción no sólo depende del
 morfotipo de mecanorreceptor presente en dichos tejidos sino también de las propiedades de las
 neuronas sensitivas primarias y las fibras sensitivas asociadas a ellos, así como de su proyección
 sobre el asta posterior de la médula espinal. Este artículo resume las bases morfológicas de la
 propiocepción a nivel del sistema nervioso periférico, analizando: a) las neuronas sensitivas
 primarias propioceptivas; b) los tipos de fibras nerviosas sensitivas que llegan a los propioceptores;
 c) la inervación sensitiva de articulaciones y músculos; d) los morfotipos de mecanorreceptores
 asociados a la propiocepción; e) los datos recientes obtenidos a partir de animales deficientes en
 factores clave para el desarrollo del sistema propioceptor.

The distribution of nerve fibres immunoreactive for vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and dopamine β-hydroxylase in the normal equine larynx

The autonomic innervation of the mammalian respiratory system is complex, and involves a wide variety of peptide and non-peptide neurotransmitters which will have an important role in normal laryngeal function and the response to disease. This innervation has been partially described in the horse airway and lung, but there is no information on the equine larynx. This paper describes the expression and distribution of nerve fibres immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and the adrenergic enzymatic marker dopamine β-hydroxylase (DBH) in the mucosa of the equine larynx. The overall relative density of nerve fibres immunoreactive for the different antigens wasVIP >>CGRP>SP>>DBH. There were differences in the distribution of nerve fibre types, although each antigen was found in nerve fibres adjacent to blood vessels and mucous glands.VIP -like immunoreactivity (VIP -Li) was particularly extensive in association with mucous glands. SP - and CGRP -like immunoreactivity (SP -Li, CGRP -Li) were also seen close to the epithelium, with occasional nerve fibres coursing beneath and between the epithelial cells. Fragments of SP -Li and CGRP -Li fibres were also present in large nerve fibre bundles and ganglionic cell clusters, but not in the neurons themselves. The density of nerve fibres immunoreactive for DBH was very low and restricted to blood vessels and mucous glands. There was marked variation in the density of nerve fibres at the different sites, with the greatest density, particularly for VIP, over the arytenoid cartilage. Immunoreactive nerve fibres were less plentiful over the epiglottis, and the density of all types of nerve fibres was low over the cricoid cartilage. Overall VIP -Li nerve fibres were the most plentiful.

Neurochemical Characterization of the Vestibular Nerves in Women with Vulvar Vestibulitis Syndrome

Women with vulvar vestibulitis syndrome (VVS) have a distinct burning pain provoked by almost any stimuli in the area around the vaginal introitus. In a previous study we observed an increased number of intraepithelial free nerve endings in women with VVS. The aim of the present study was to neurochemically characterize the superficial nerves in the vulvar vestibular mucosa of women with VVS. Immunohistochemical methods were used to detect neuropeptides normally found in various types of nerve fibers. Calcitonin gene-related peptide, which is known to exist in nociceptive afferent nerves, was the only neuropeptide detected in the superficial nerves of the vestibular mucosa. These findings confirm our previous theory that the free nerve endings within the epithelium are nociceptors.

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.

New developments in the diagnosis of diabetic neuropathy

The development of new treatments to slow or arrest the progression of diabetic polyneuropathy (DPN) has increased the importance of the early and accurate identification of this complication. It is likely that effective intervention will be possible only during the subclinical or early phase of dysfunction. Accurate diagnosis of DPN is a formidable task because of the diversity of presentations, involvement of different nerve fiber types, and the common dissociation of symptoms from objective measures of neural function. Several diagnostic tools are available or in development, each with strengths and limitations. Electrophysiology is a sensitive, objective, and targeted measure of DPN, but it reflects, almost exclusively, the activity of large-caliber, myelinated axons. Newly refined skin-punch biopsy procedures use morphometric and immunohistochemical methods to examine thinly myelinated and unmyelinated nerve fibers. The integrity, density, and distribution of these fibers may provide a sensitive index of small-fiber distal axonopathy. Improvements in quantitative sensory testing include better control of stimulation characteristics and the use of computer-assisted testing algorithms (e.g., CASE IV), as well as the ability to examine a distal to proximal gradient of sensation (Physitemp NTE-2a). Composite scales, which combine the assessment of signs, symptoms, electrophysiology, and specific quantitative sensory measures, have also been proposed and, in some cases, validated. The existing diagnostic tools and newly emerging methods provide a battery of tests that can be used to assess multiple aspects of neural function and increase sensitivity to detect the onset and progression of DPN.

Innervation of blood vessels in the vomeronasal complex of the rat

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for α-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.

Differential onset of median nerve block: randomized, double-blind comparison of mepivacaine and bupivacaine in healthy volunteers

We have compared the delay in onset of 1% mepivacaine and 0.33% bupivacaine in different nerve fibre types in 10 volunteers undergoing median nerve blocks, in a randomized, double-blind, crossover study. Hot, cold, pinprick and light touch sensations, compound motor action potentials (CMAP), sensory nerve action potentials (SNAP) and skin temperature were recorded at 2-min intervals. Hot, cold, pinprick, light touch sensations, SNAP and CMAP were significantly inhibited, and skin temperature was significantly increased after administration of both agents. The first noticeable reduction in cold sensation was detected later after bupivacaine compared with mepivacaine, but after a delay similar to that of other nerve functions. Bupivacaine and mepivacaine inhibited SNAP and CMAP with a similar time delay to steady-state. Bupivacaine produced steady-state inhibition of hot and cold sensations significantly later than mepivacaine; nevertheless, the sequence that sensory modalities failed, with few exceptions, and the extent of anaesthesia at 40 min were similar for both agents. Our technique provides a novel, multi-modal method of comparing local anaesthetics and related agents over time.

Subclinical neuropathy in type I diabetic children

Objectives: Small and large, somatic and autonomic nerve fibre functions were neurophysiologically evaluated in 33 asymptomatic neurologically free type 1 diabetic children and 69 age-matched healthy controls. Methods: The evaluation of large and small somatic nerve fibre function was performed by conventional nerve conduction studies, thermal specific and thermal pain sensitivity tests, as well as autonomic nerve fibre functions by sympathetic skin response and R–R interval variation assessment. Results: A significant difference was established between the healthy and the diabetic group. Neurophysiologically determined subclinical neuropathy was found in 87% of type 1 diabetic children. The majority of abnormal recordings were found on the lower limbs. The dysfunction of the somatic motor large nerve fibre type in the lower limbs was altered in 57% of patients, somatic sensory large in 39%, somatic sensory small in 45%, and sympathetic in 45%. The leading abnormal measure was a delayed sympathetic skin response on the foot (42% of diabetic children) followed by a reduced amplitude of sural nerve action potential (36%). The whole spectrum of recordings showed scattered involvement of nerve functions. There was no selective susceptibility of nerve fibre types exposed to a noxious factor. Conclusion: A complex neurophysiological assessment, including standard nerve conduction studies as well as psychophysical examination and autonomic nerve function tests, evaluating the function of small and large nerve fibres, is recommended for evaluating the subclinical neuropathy in asymptomatic type I diabetic children.

Nerve ingrowth into diseased intervertebral disc in chronic back pain

In the healthy back only the outer third of the annulus fibrosus of the intervertebral disc is innervated. Nerve ingrowth deeper into diseased intervertebral disc has been reported, but how common this feature is and whether it is associated with chronic pain are unknown. We examined nerve growth into the intervertebral disc in the pathogenesis of chronic low back pain. We collected 46 samples of intervertebral discs from 38 patients during spinal fusion for chronic back pain. 30 samples were from pain levels clinically established by discography and 16 samples were from adjacent vertebral levels with no pain. We obtained 34 control samples of intervertebral disc from previously healthy individuals with normal histology within 8 h of recorded death. We used standard immunohistochemical techniques to test for a general nerve marker, a nociceptive neurotransmitter (substance P), and a protein expressed during axonogenesis (growth-associated protein 43 [GAP43]). We identified nerve fibres in the outer third of the annulus fibrosus in 48 (60%) of the 80 samples of intervertebral discs. Nerves were restricted to the outer or middle third of the annulus fibrosus in the 34 control samples. Among the patients with chronic low back pain, nerves extended into the inner third of the annulus fibrosus and into the nucleus pulposus in 21 (46%) and ten (22%) samples, respectively. Nerves usually accompanied blood vessels, but in 14 of the samples from back-pain patients, isolated nerve fibres were seen in the discal matrix. Both types of nerve fibres expressed substance P, but only non-vessel-associated fibres expressed GAP43. Deep nerve ingrowth into the inner third of the annulus fibrosus, the nucleus pulposus, or both was seen in four (25%) of 16 biopsy samples from non-pain levels and in 17 (57%) samples from pain levels. Of the 16 paired samples from both pain and non-pain levels, five pain-level samples and one non-pain-level sample showed deep nerve ingrowth. Our finding of isolated nerve fibres that express substance P deep within diseased intervertebral discs and their association with pain suggests an important role for nerve growth into the intervertebral disc in the pathogenesis of chronic low back pain.

A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle--dependent changes in mast cell--nerve fiber contacts in murine skin.

Close contacts between mast cells (MC) and nerve fibers have previously been demonstrated in normal and inflamed skin by light and electron microscopy. A key step for any study in MC-nerve interactions in situ is to simultaneously visualize both communication partners, preferably with the option of double labelling the nerve fibers. For this purpose, we developed the following triple-staining technique. After paraformaldehyde-picric acid perfusion fixation, cryostat sections of back skin from C57BL/6 mice were incubated with a primary rat monoclonal antibody to substance P (SP), followed by incubation with a secondary goat-anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then applied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG. MCs were visualized by incubation with AMCA-labelled avidin, or (for a more convenient quantification of close MC-nerve fiber contacts) with a mixture of TRITC- and FITC-labelled avidins. Using this simple, novel covisualization method, we were able to show that MC-nerve associations in mouse skin are, contrary to previous suggestions, highly selective for nerve fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MCs preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR double-labelled nerve fibers. Compared with telogen values, there was a significant increase in the number of close contacts between MCs and tyrosine hydroxylase-IR fibers during late anagen, and between MCs and peptide histidine-methionine-IR and choline acetyl transferase-IR fibers during catagen.