Evaluation of vestibular biopsy features in patients affected by fibromyalgia, by vulvodynia or by their association.

OBJECTIVE: To evaluate the histologic features of vestibular biopsies from patients affected by fibro myalgia (FM), or vulvodynia (VD), or the their association (FM-VD) in order to facilitate differential diagnosis among conditions that present sexual pain with similar clinical characteristics. STUDY DESIGN: Fourty-four women already diagnosed with FM were recruited to evaluate the presence of sexual pain not owing to FM. Fourteen women affected by sexual pain of unknown origin who came to our department requesting treatment were also recruited. All subjects were interviewed regarding their history of pain and examined in order to exclude vaginal conditions. Sexual pain did not show the characteristics of VD in 18 FM women; in the remaining 22 women VD resulted as associated with FM. All fourteen self-referred women were diagnosed with VD. All subjects underwent a posterior vestibular biopsy at the fourchette under local anesthesia. Tissue specimens were processed for histologic examination and immunostained for S-100protein and CD34. Statistical analysis was performed with the Pearson's Chisquare test. RESULTS: Data analysis showed a statistically significant prevalence of inflammation in the VD group. Analysis of the histologic features showed that the concomitant presence of inflammation, nerve bundles, and fibrosis (often mild) is prevalent in VD. Fibrosis is highly frequent and often moderate/severe in FM and it is rarely associated to inflammation and nerve bundles. FM-VD women show intermediate grading. CONCLUSIONS: Our findings show different histologic characteristics in vestibular biopsy in patients affected by Fibro Myalgia, by Vulvodynia or by their association that could be useful to facilitate the differential diagnosis between conditions of sexual pain with similar clinical characteristics.

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells.

CONCLUSION: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. OBJECTIVES: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. MATERIALS AND METHODS: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. RESULTS: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

Aquaporin-2 in the human endolymphatic sac.

OBJECTIVE: To detect and localize aquaporin-2 (AQP-2), a water channel regulated by the antidiuretic hormone, in human endolymphatic sac. MATERIAL AND METHODS: Human endolymphatic sacs were sampled during removal of vestibular schwannomas via a translabyrinthine approach. Samples were immediately fixed in 10% formalin (24 h) and embedded in paraffin; in situ hybridization and immunohistochemistry were performed with an AQP-2-specific probe and a polyclonal antibody. RESULTS: Both AQP-2 mRNA and protein were detected in the epithelium of the endolymphatic sac. AQP-2 immunostaining was mainly cytoplasmic, suggesting that most AQP-2 was located in intracellular pools. CONCLUSIONS: In the endolymphatic sac, AQP-2 probably participates in the homeostasis of endolymph; the possibility of reducing the volume of endolymph by inhibiting its expression and membranous insertion using an antidiuretic hormone inhibitor represents a new therapeutic approach for the treatment of Ménière's disease.

Alteration of E-cadherin and beta-catenin in mouse vestibular epithelia during induction of apoptosis.

The aim of this study was to examine if adhesion molecules had relation with degeneration and regeneration processes of mammalian vestibular epithelia. The distribution of E-cadherin and beta-catenin was immunohistochemically examined in normal and aminoglycoside-treated utricles of mice. E-cadherin and beta-catenin linearly expressed between epithelial cells in normal specimens. Aminoglycoside injury resulted in temporal alteration in distribution of these molecules with induction of apoptosis in hair cells. Degradation of both molecules was widely observed in vestibular epithelia, while some supporting cells exhibited accumulation of beta-catenin. After completion of induction of apoptosis, expression of these adhesion molecules was normal in distribution. These findings suggest that the E-cadherin-beta-catenin complex plays roles in degeneration and subsequent repair processes in vestibular epithelia affected by aminoglycosides.

PTEN is not altered in sporadic vestibular schwannomas.

AIMS: To investigate the role of the tumour suppressor gene PTEN in the tumorigenesis and growth of sporadic vestibular schwannomas, and to characterize the cellular distribution of the PTEN protein in relation to the MIB-1 proliferation index in these tumour. METHODS AND RESULTS: Immunoexpression of the PTEN protein was observed within the neoplastic Schwann cells in 21 out of 30 sporadic schwannomas examined (70%). PTEN expression was consistently stronger in Antoni A areas than in Antoni B areas. High levels of PTEN immmunoexpression in schwannomas were associated with an increased MIB-1 labelling index. Occasionally, vascular endothelial cells also showed PTEN immunoreaction. By polymerase chain reaction-single strand conformation polymorphism screening, no mutations were found in the complete protein coding region of the PTEN gene. CONCLUSIONS: The PTEN tumour suppressor gene is expressed in the majority of sporadic schwannomas. The maintained expression of the PTEN protein, together with the lack of detectable mutations in this gene, suggests that the function of the PTEN tumour suppressor gene is not altered in sporadic vestibular schwannomas.

Connexin 26 distribution in gap junctions between melanocytes in the human vestibular dark cell area.

We have previously demonstrated the presence of gap junctions between melanocytes in the human vestibular organ and have speculated that melanocytes function in maintaining the homeostasis of the microenvironment of the inner ear. The purpose of the present study was to characterize the expression and ultrastructural localization of connexin (Cx) protein in melanocytes of the human vestibular organs. Surgical material was obtained from patients operated on for vestibular schwannoma and was processed for light microscopy, confocal laser scanning microscopy, conventional TEM, and immuno TEM. The specimens were labeled with anti-Cx26, Cx32, and Cx43 antibodies and examined by light microscopy. Specimens were also labeled with anti-Cx26 antibody and examined by laser microscopy and immuno-TEM methods. The specimens examined in this study were mainly dark cell areas from the human vestibular organ, whose epithelial and subepithelial layers are rich in melanocytes. Light-microscopic immunohistochemical studies showed positive labeling for Cx26 protein between subepithelial melanocytes, and Cx32 was also detected. Use of anti-Cx26 antibody and confocal laser scanning microscopy revealed high levels of Cx26 around the subepithelial melanocytes. Post-embedding immuno-gold transmission electron microscopy showed significant aggregation of gold particles (33.97 +/- 8.01% of total gold particles) around the gap junctions of the subepithelial melanocytes. The results of this study indicated that melanocytes are connected through gap junctions that mainly contain Cx26. This suggested that the melanocytes in the human vestibular organ may play a role in transporting material between the endolymph and perilymph.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF-receptors Flt-1 and KDR/Flk-1 in the vestibule of guinea pigs.

Vascular endothelial growth factor (VEGF), known as an endothelial cell-specific mitogen, has been reported to be linked also to the NO/cGMP-pathway, which has been notified in the inner ear. Up to now, VEGF has not yet been described in the inner ear. We performed immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF-receptors Flt-1 and KDR/Flk-1 on paraffin-sections of temporal bones from guinea pigs (n=5). Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in a subpopulation of vestibular ganglion cells. VEGF could be found also in the endothelium of blood vessels, in fibrocytes of the lamina propria and in the neuroepithelium. Strong immuno-labelling to Flt-1 was evident in nerve fibres, vascular endothelium and in the neuroepithelium. Fibrocytes, endothelium of blood vessels, supporting cells and calyces in the sensory epithelium revealed immunoreactivity to KDR/Flk-1. These findings give evidence that VEGF, Flt-1 and KDR/Flk-1 are constitutively expressed in the vestibule.

Involvement of insulin-like growth factor-I in inner ear organogenesis and regeneration.

The verterbrate inner ear is an excellent model system to study signalling mechanisms in embryonic development. During the last years, insulin-like growth factor-I (IGF-I) has attracted attention in relation to the regulation of inner ear ontogenesis. IGF-I and its high-affinity tyrosine-kinase receptor are expressed during early stages of inner ear development. IGF-I is a powerful mitogen for the otic vesicle, where it stimulates cell-division and mitogenic signalling cascades. Later in development, IGF-I also promotes survival and neurogenesis of the otic neurones in the cochleovestibular ganglion (CVG). The actions of IGF-I are associated with the generation of lipidic messengers and the activation of Raf kinase, which results in the rapid induction of the expression of the proliferative cell nuclear antigen (PCNA) and the nuclear proto-oncogenes c-fos and c-jun. Regulation of organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. A model is proposed where this balance is the consequence of the action of IGF-I and NGF, which converge in Raf activation or suppression. The combinatorial expression of jun and Fos family members in particular domains of the otic vesicle would be the final result of such cascade. Some of these mechanisms may be also implicated in otic regeneration.

Apoptosis in the human inner ear. Detection by in situ end-labeling of fragmented DNA and correlation with other markers.

The aim of this study was to obtain baseline data on the recently described special form of single cell death, apoptosis, in normal human inner ears. For this purpose, in situ end-labeling of the fragmented DNA was applied, in conjunction with apoptosis-related markers, to detect cellular elements showing programmed cell death in decalcified and paraffin-embedded tissues. Over 20 specimens were analyzed which were obtained from autopsy cases with no history of acoustic lesions confirmed by histopathology. Based on staining results, we saw no apoptotic signs in the majority of normal adult inner ears. An apoptotic cell captured in the Reissner's membrane of the cochlea from an old patient may, however, indicate an age-related subtle cell loss with the process of apoptosis. Nevertheless, the fact that more apoptosis was not found in our cases suggests that this phenomenon does not contribute significantly to the tissue homeostasis in the adult inner ear under normal conditions. These data are in accordance with our immunohistochemical findings on the p53 nucleoprotein, and proliferating cell nuclear antigen expression since there was no staining in any of the cellular elements, including the mesenchymal cells. This reflects a stationary and stable condition of cells of the vestibular and the cochlear structures, probably to maintain their integrity and the fine sensory functions. As opposed to the above findings, during inner ear development, the epithelial cells lining the cochlear lumen, the ossifying cartilage of the temporal bone, and the mesenchymal cells show different degrees of proliferation in combination with single cell death as signs of maturation of the vestibular and the cochlear apparatus. In addition, apoptosis has been demonstrated in cells of the cochlear stria vascularis from an adult patient treated with high doses of cisplatin, vinblastine and bleomycin prior to death. Furthermore, a wide range of apoptosis could be induced experimentally in a normal ear by an external perfusion of actinomycin D (ActD), which is known to produce programmed cell death in many cell types of different origins. The potential role of cytostatic agents in the apoptotic process of the inner ear needs, however, to be confirmed in large-scale specimens from patients treated with genotoxins. The fact, however, that apoptotic cells are also seen in association with ActD indicates that the fine sensory structure of the cochlea may also be a target for certain chemotherapeutic agents when administered in high doses.

Rab3A immunolocalization in the mammalian vestibular end-organs during development and comparison with synaptophysin expression.

Rab proteins are essential for membrane vesicle docking and fusion and for transport vesicle formation at the presynaptic membrane, a step in the release of neurotransmitters. The vestibular sensory epithelia contain three types of synapses: afferent terminals, efferent endings and possible synaptic contacts between the apex of the afferent nerve calyces and the sensory cells. We report an immunocytochemical codetection of rab3A and synaptophysin in the vestibular end-organs of mouse, between fetal day 14 and adult, and of rat during the postnatal development. During mouse fetal development, rab3A appeared in afferent neurites on F16, and in sensory cells on F19. This was respectively two and five days later than the appearance of synaptophysin-IR in the same compartments. During the late postnatal development and in the adult sensory epithelia, rab3A and synaptophysin were strongly detected in nerve terminals of efferent and possibly afferent nature and in the upper part of the nerve calyces. The presence of rab3A in the nerve calyces is consistent with the putative secretory function of the calyx. In addition, rab3A immunostaining was also present in the sensory cells together with a faint synaptophysin-IR, that had not been described in previous reports [Scarfone, E., Demêmes, D. and Sans, A. J. Neurosci., 11 (1991) 1173-1181.]. The presence of these two proteins in the sensory cells supports the existence of a synaptic vesicle cycle in these cells.

Existence of voltage-dependent Ca2+ channels in vestibular dark cells: cytochemical and whole-cell patch-clamp studies.

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.

Receptors for glucocorticoids in the human inner ear.

Glucocorticoid receptors were detected in the human inner ear. The highest concentration of glucocorticoid receptor protein was measured by enzyme-linked immunosorbent assay in the spiral ligament tissues; the lowest concentration of glucocorticoid receptors was measured in the macula of the saccule. The demonstration of the presence of glucocorticoid receptors in human Inner ear tissues provides a basis to consider the direct effects of glucocorticoid action on select inner ear cells, rather than assuming a systemic antiinflammatory or immunosuppressive effect during the therapeutic treatment of patients with given inner ear disorders.

Differential cellular distribution of glutamate and glutamine in the rat vestibular endorgans: an immunocytochemical study.

The cellular and subcellular localization of glutamate and glutamine in the rat vestibular endorgans was studied by means of postembedding immunocytochemistry. Glutamate immunoreactivity was preferentially distributed in the hair cells, whereas glutamine immunoreactivity was enriched in supporting cells. This points to a metabolic compartmentation similar to that found in glutamatergic nerve terminals and adjacent glial processes in the central nervous system. The present immunocytochemical results are consistent with the existence of a glutamate-glutamine cycle in the vestibular sensory epithelium. Our data are also in agreement with a transmitter role of glutamate in both types of hair cell, although a vesicular enrichment of glutamate in these cells remains to be demonstrated.

Distribution of alpha-ANP in the cochlea and the vestibular organs.

The distribution of rat alpha-atrial natriuretic polypeptide (ANP) was studied in the adult inner ear of the Sprague-Dawley rat using immunohistochemical methods. Immunostaining, in particular of the subepithelial stroma cells of the spiral ligament, the spiral prominence and below neuroepithelial areas of all five vestibular organs, was identified. We hypothesize that alpha-ANP is involved in perilymphatic dynamics rather than generating endolymph. The strong staining for alpha-ANP of outer hair cells indicates specific mechanisms for volume control as compared with inner hair cells and vestibular hair cells which all lack this immunostaining. The functional significance of the specific staining of nerve calyces in only the three cristae ampullaris but not in the two maculae remains to be clarified. The positivity of epithelial cells in the endolymphatic sac supports a previous hypothesis which looks upon this organ as a pressure regulation system in the inner ear.

Immunohistochemical localization of brain type glucose transporter in mammalian inner ears: comparison of developmental and adult stages.

Inner ears from five mammalian genera were examined immunohistochemically with a rabbit polyclonal antiserum against the brain type glucose transporter (GLUT1). Vascular endothelial cells distributed widely in soft tissues of the cochlea and vestibular system in all five genera showed uniform immunostaining. The basal cell layer of the stria vascularis also contained GLUT1 in all genera, and in the guinea pig, the strial marginal cells reacted as well. GLUT1 was expressed in satellite cells surrounding spiral ganglion neurons but only in the gerbil and cat. In the developing inner ear of the gerbil, endothelial cells expressed GLUT1 at 2 days after birth, the earliest stage examined. Immunoreactive transporter also was detected at this time in cells lying under strial marginal cells and interpreted as immature basal cells. Satellite cells acquired affinity for GLUT1 antibody between days 12 and 16 after birth. The expression of GLUT1 by the various cell types correlates well with their structural and functional maturation. GLUT1 apparently plays a role in glucose transport in the inner ear where it mediates efflux from blood vessels into perilymph. It also appears to facilitate uptake of glucose by the stria vascularis from interstitial fluid via the basal cell layer and, in some species, by spiral ganglion neurons through satellite cells.

Cell and molecular anatomy of nicotinic acetylcholine receptor subunits and calcitonin gene-related peptide in the rat vestibular system.

In this report we demonstrate the pattern of calcitonin gene-related peptide (CGRP) mRNA and immunoreactivity in the central and peripheral vestibular system of the rat, using a CGRP cRNA probe and a polyclonal CGRP antiserum. We present evidence that somata in all regions of efferent vestibular neurons contain CGRP based on the correspondence between in situ hybridization (mRNA) and immunohistochemistry (mRNA translation product). CGRP immunohistochemistry (CGRPi) and in situ hybridization confirm that CGRPi axons and terminals present in the vestibular neuroepithelium are efferent in origin. Immunoelectron microscopy revealed an extensive innervation of the afferent vestibular pathway by CGRPi terminals that was not limited to the primary afferent chalice, as previously reported by Tanaka et al. (Brain Res 1989;504:31-5). An efferent neuromodulatory role of CGRP can be inferred from the distribution of terminals found on the primary afferent fibers, and type I and type II hair cells. In addition, we present evidence that nicotinic acetylcholine receptor (nAChR) subunit mRNA is expressed by primary afferent cell bodies. On the basis of these data, a hypothetical molecular mechanism of vestibular efferent modulation of the primary afferent pathway is proposed.