Marita Hilliges

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.

SummarySections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.

Increased Intraepithelial Innervation in Women with Vulvar Vestibulitis Syndrome

Women with vulvar vestibulitis syndrome (VVS) suffer from severe pain and discomfort in the area around the introitus at almost any stimulus that causes pressure within the vestibule. In spite of the severe sensory symptoms present in these women, the influence of the peripheral nerves in the vulvar vestibulum has not been clarified before. In this study the nerve supply in the vestibular mucosa in women with VVS and in healthy women free from vulvar symptoms has been revealed by PGP 9.5 immunohistochemistry. The results show a significant increase in the number of intraepithelial nerve endings in women with VVS, indicating an alteration in the nerve supply in the afflicted area.

Psychophysical evidence of nociceptor sensitization in vulvar vestibulitis syndrome

&NA; Vulvar vestibulitis syndrome (VVS) is a long lasting disorder of superficial dyspareunia in young women. Quantitative sensory testing, including mechanical and temperature pain thresholds and warm/cold difference limen (WCL), was performed in the vestibular mucosa in 22 women (mean age 25.0 years) with vestibulitis and 20 control subjects (mean age 25.6 years). The tests were carried out on days 7–11 of the menstrual cycle. Patients had allodynia to mechanical testing with von Frey filaments, 14.3±3.1 mN in the symptomatic posterior area as compared with 158±33.5 mN in healthy subjects, P<0.0001. The pain threshold to heat was 38.6±0.6°C in patients and 43.8±0.8°C in controls, P<0.0001. In addition, pain threshold to cold was 21.6±1.2°C in patients whereas cooling down to 6°C was usually not painful in controls. WCL was 4.9±0.5°C in patients and 9.6±1.5°C in healthy subjects, P<0.01. The results are compatible with the hypothesis that patients with VVS have an increased innervation and/or sensitization of thermoreceptors and nociceptors in their vestibular mucosa.

Abnormal Function of C-Fibers in Patients with Diabetic Neuropathy

The mechanisms underlying the development of painful and nonpainful neuropathy associated with diabetes mellitus are unclear. We have obtained microneurographic recordings from unmyelinated fibers in eight patients with diabetes mellitus, five with painful neuropathy, and three with neuropathy without pain. All eight patients had large-fiber neuropathy, and seven patients had pathological thermal thresholds in their feet, indicating the involvement of small-caliber nerve fibers. A total of 163 C-fibers were recorded at knee level from the common peroneal nerve in the patients (36–67 years old), and these were compared with 77 C-fibers from healthy controls (41–64 years old). The ratio of mechano-responsive to mechano-insensitive nociceptors was ∼2:1 in the healthy controls, whereas in the patients, it was 1:2. In patients, a fairly large percentage of characterized fibers (12.5% in nonpainful and 18.9% in painful neuropathy) resembled mechano-responsive nociceptors that had lost their mechanical and heat responsiveness. Such fibers were rarely encountered in age-matched controls (3.2%). Afferent fibers with spontaneous activity or mechanical sensitization were found in both patient groups. We conclude that small-fiber neuropathy in diabetes affects receptive properties of nociceptors that leads to an impairment of mechano-responsive nociceptors.

Innervation of the human vaginal mucosa as revealed by PGP 9.5 immunohistochemistry.

In order to obtain a description of the innervation of the vaginal wall we employed an antiserum against the general neuronal marker, protein gene product 9.5, on normal human vaginal mucosa. Specimens were taken from the anterior and posterior fornices, from the anterior vaginal wall at the bladder neck level and from the introitus vaginae region, and then processed for indirect immunohistochemistry. All regions studied revealed a profound innervation, although regional differences were noted. The more distal areas of the vaginal wall had more nerve fibers compared to the more proximal parts. Also, biopsies from the anterior wall generally were more densely innervated than the posterior wall. Some large nerve coils were observed in lamina propria of the anterior wall as well as gatherings of thick-walled medium-sized blood vessels. Free intraepithelial nerve endings were only detected in the introitus vaginae region. These fibers were very thin, always varicose and could be observed just a few cell layers from the surface. In this part of the vagina, protein gene product 9.5 antibodies also stained cells within the basal parts of the epithelium. These cells were also neurone-specific enolase positive and resembled, from a morphological point of view, Merkel cells.

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.

Active "itch fibers" in chronic pruritus.

An itch-specific neuronal pathway was recently discovered in healthy humans and animals. Here the authors report that activity in this specific pathway coincides with itch under pathophysiologic conditions in a patient with chronic pruritus. Microneurographic recordings from the symptomatic area revealed spontaneous activity in six single C-fiber afferents that had the characteristic features of “itch fibers.” Itch may be caused by activity in a specific subpopulation of C-fiber afferents.

Electrically evoked neuropeptide release and neurogenic inflammation differ between rat and human skin

1 Protein extravasation and vasodilatation can be induced by neuropeptides released from nociceptive afferents (neurogenic inflammation). We measured electrically evoked neuropeptide release and concomitant protein extravasation in human and rat skin using intradermal microdialysis. 2 Plasmapheresis capillaries were inserted intradermally at a length of 1.5 cm in the volar forearm of human subjects or abdominal skin of rats. Capillaries were perfused with Ringer solution at a flow rate of 2.5 or 1.6 μl min−1. After a baseline period of 60 min capillaries were stimulated electrically (1 Hz, 80 mA, 0.5 ms or 4 Hz, 30 mA, 0.5 ms) for 30 min using a surface electrode directly above the capillaries and a stainless‐steel wire inserted in the capillaries. Total protein concentration was assessed photometrically and calcitonin gene‐related peptide (CGRP) and substance P (SP) concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). 3 In rat skin, electrical stimulation increased CGRP and total protein concentration in the dialysate. SP measurements showed a larger variance but only for the 1 Hz stimulation was the increased release significant. 4 In human skin, electrical stimulation provoked a large flare reaction and at a frequency of 4 Hz both CGRP and SP concentrations increased significantly. In spite of the large flare reactions no protein extravasation was induced, which suggests major species differences. 5 It will be of interest to investigate whether the lack of neurogenic protein extravasation is also valid under pathophysiological conditions.

Neural Signal Processing: The Underestimated Contribution of Peripheral Human C-Fibers

The microneurography technique was used to analyze use-dependent frequency modulation of action potential (AP) trains in human nociceptive peripheral nerves. Fifty-one single C-afferent units (31 mechano-responsive, 20 mechano-insensitive) were recorded from cutaneous fascicles of the peroneal nerve in awake human subjects. Trains of two and four suprathreshold electrical stimuli at interstimulus intervals of 20 and 50 msec were applied to the receptive fields of single identified nociceptive units at varying repetition rates. The output frequency (interspike interval) recorded at knee level was compared with the input frequency (interstimulus interval) at different levels of accumulated neural accommodation. At low levels of use-dependent accommodation (measured as conduction velocity slowing of the first action potential in a train), intervals between spikes increased during conduction along the nerve. At increasing levels of neural accommodation, intervals decreased because of a relative supernormal period (SNP) and asymptotically approached the minimum “entrainment” interval of the nerve fiber (11 ± 1.4 msec) corresponding to a maximum instantaneous discharge frequency (up to 190 Hz). For neural coding, this pattern of frequency decrease at low activity levels and frequency increase at high levels serves as a mechanism of peripheral contrast enhancement. The entrainment interval is a good minimum estimate for the duration of the refractory period of human C-fibers. At a given degree of neural accommodation, all afferent C-units exhibit a uniform pattern of aftereffects, independent of fiber class. The receptive class of a fiber only determines its susceptibility to accommodate. Thus, the time course of aftereffects and existence or absence of an SNP is fully explained by the amount of preexisting accommodation.

ATP responses in human C nociceptors.

&NA; Microelectrode recordings of impulse activity in nociceptive C fibres were performed in cutaneous fascicles of the peroneal nerve at the knee level in healthy human subjects. Mechano‐heat responsive C units (CMH), mechano‐insensitive but heat‐responsive (CH) as well as mechano‐insensitive and heat‐insensitive C units (CMiHi) were identified. A subgroup of the mechano‐insensitive units was readily activated by histamine. We studied the responsiveness of these nociceptor classes to injection of 20 &mgr;l 5 mM adenosintriphosphate (ATP) using saline injections as control. Because of mechanical distension during injection, which typically activates mechano‐responsive C fibres, interest was focused on responsiveness to ATP after withdrawal of the injection needle. Post‐injection responses were observed in 17/27 (63%) mechano‐responsive units and in 14/22 (64%) mechano‐insensitive units. Excitation by ATP occurred in 9/11 CH units and in 5/11 CMiHi units. ATP responsive units were found both within the histamine‐responsive and the histamine‐insensitive group of mechano‐insensitive fibres. ATP responses appeared with a delay of 0–180 s after completion of injection; responses were most pronounced during the first 1–3 min of activation, and irregular ongoing activity was observed for up to 10 or even 20 min. ATP responses were dose‐dependent, concentrations lower than 5 mM gave weaker responses. No heat or mechanical sensitisation was observed in any of the major fibre classes. In conclusion, we have shown that ATP injections at high concentrations activate C‐nociceptors in healthy human skin, without preference for mechano‐responsive or mechano‐insensitive units. ATP did not sensitise human C fibres for mechanical or heat stimuli. We discuss how various mechanisms might contribute to the observed responses to ATP.