Primary cultures of human vestibular schwannoma: selective growth of schwannoma cells.

OBJECTIVE: To establish primary vestibular schwannoma (VS) cultures that selectively favor the growth of schwannoma cells. BACKGROUND: The lack of a suitable in vitro model of human VS cells has directly limited the progress of research on tumorigenesis and therapy. The problems of establishing pure VS culture include control of fibroblast proliferation. Current efforts to extend VS cell life span using viral oncogenes, by conferring the ability to proliferate in vitro, will yield cells intrinsically different from in vivo VS tumors. Much more desirable is the ability to culture VS cells without cellular transformation. METHODS: Tumor specimens from 17 patients were processed for cell culture and grown at 37 degrees C with 5% CO2 and 100% humidity. Key modifications limiting fibroblast proliferation included using selective medium without L-valine, supplemented by Nu-Serum for at least a week; the use of cytosine arabinoside to kill contaminating fibroblasts; and using the Dulbecco modified medium, supplemented with brain-derived neurotrophic factor and 10% fetal calf serum after the initial serum-free period. RESULTS: Twelve of 17 VS were successfully cultured. The presence of schwannoma cells and the absence of fibroblasts were confirmed immunohistochemically using S100 and CD90 markers, respectively. Scanning and transmission electron microscopy demonstrated typical spindle-shaped cells and the presence of "fibrous long-spacing collagen." CONCLUSION: We describe a method for obtaining short-term, essentially fibroblast-free, primary VS cultures. Such pure VS cultures, retaining in vivo characteristics, are extremely useful as an in vitro model to study the pathobiology of schwannoma cells.

Acoustic neuromas and meningiomas. Histopathological aspect: a post mortem study on temporal bones.

The histopathological changes in the temporal bones of three deceased donors individuals, two with concomitant acoustic tumors and one with concomitant petroclival meningioma have been studied. The differences between neuromas and meningiomas are examined. The different clinical relevance and the different therapeutic guidelines are discussed.

[The neurodystrophic component of the structural changes in the hemispheric cerebellar cortex of patients with acoustic neurinomas]. / Neirodistroficheskii komponent strukturnykh izmenenii v kore polusharii mozzhechka u bol'nykh s nevrinomami slukhovogo nerva.

Cerebellar specimens removed in surgical treatment of acoustic nerve neurinomas were studied morphologically in 16 patients. Histochemically, morphofunctional state of the vegetative adrenergic nervous fibers and endings of the vessels and connective tissue basis of the pia mater encephali in the region of the cerebellum indicates inhibited activity of the neuromediator contour of circulation and metabolism regulation in the brain. Light and electron microscopy in the cerebellar cortex detected disorders of circulation, nervous cell dystrophy, glyocytic proliferation. Correlation was found between activity of sympathetic component of the autonomic nervous system, severity of neurodystrophic changes in the cerebellar cortex and development of pathophysiological reactions arising intraoperatively and early after operation.

The morphological differences of stereocilia and cuticular plates between type-I and type-II hair cells of human vestibular sensory epithelia.

The sensory epithelia of macula utriculi were examined by conventional and intermediate voltage transmission electron microscopy. The specimens were obtained from three cases of acoustic neurinoma who were operated on using the translabyrinthine approach. The mean diameter of the vestibular hair cell stereocilia was obtained and the cuticular plates of type-I and type-II hair cells were reconstructed three-dimensionally from the consecutive 0.5-micron-thick sections. The mean diameter of stereocilia of type-I hair cells was 488 +/- 59 nm (n = 13) and that of stereocilia of type-II hair cells was 373 +/- 21 nm (n = 14). Stereocilia of type-I hair cells numbered about 70 and those of type-II hair cells about 50. The cuticular plates of type-I hair cells were several times as thick as those of type-II hair cells. The cuticular plate of the type-I hair cell appeared to be an inverse cone and that of the type-II hair cell seemed to be a flat disc.

Acoustic neurinoma with a large organized hematoma--case report.

A 66-year-old female presented with a large organized hematoma within an acoustic neurinoma. She had suffered from diminished hearing for 20 years and had headache 1 week before presentation. Computed tomography demonstrated an inhomogeneously high density cerebellopontine angle mass, and magnetic resonance imaging showed a mass with heterogeneous intensity and gadolinium-diethylenetriaminepenta-acetic acid enhancement of only the peripheral surface of the mass and the inner parts of the internal auditory meatus. At operation the majority of the mass was soft and feature-less with a firm capsule, and a yellowish soft tumor was removed from the perimeatal area. Histological examination showed the mass was an acoustic neurinoma with a large organized hematoma. Extensive hemorrhage from an abnormal vascularity in the tumor had repeated followed by granulomatous organization.

Histopathology of the peripheral vestibular system in small vestibular schwannomas.

Gadolinium-enhanced magnetic resonance imaging can be used to detect small vestibular schwannomas/acoustic neuromas. Early detection raises the question of the necessity of their surgical removal. Do all tumors induce lesions in the vestibule and to what extent? We thus investigated the ultrastructure of peripheral vestibular systems in grades I and II schwannomas. Vestibular tissues were fixed as soon as they were removed during the resection of tumors, by the translabyrinthine approach, and then processed for transmission electron microscope observations. In neurosensory epithelia, hair cells lost stereocilia, whereas cuticular plates disaggregated. The cytoplasm of hair cells degenerated in either a dense or vacuolated manner, and cytoplasmic blisters extended into the endolymph. In some cases, supporting cells extended processes covering the apical surface of hair cells. Nerve fibers massively disappeared from epithelia, only few nerve fibers contacted type I and type II hair cells, and both afferent and efferent terminals were abnormal. In vestibular nerves, axons degenerated, and myelin sheaths disaggregated. Glycogen was present in both intracellular and extracellular spaces. Luse bodies associated with collagen bundles were found between fibers. Scarpa ganglion neurons contained lysosomes/lipofuscin granules and vacuoles. Tumor cells were found in both the ganglion and the vestibular nerve. Thus small tumors induce extensive degeneration of vestibular tissues. The various hallmarks of schwannomas are already present in small acoustic neuromas. Moreover, different types of degeneration of hair cells and neurons were observed, together with the covering of hair cells by supporting cells and the accumulation of glycogen in the vestibular nerve.

Angiogenesis from the eighth cranial nerve to vestibular schwannomas.

Vascularization between vestibular schwannomas and the adjacent eighth cranial nerve was examined in order to elucidate the angiogenic effect of these tumours. Specimens from 12 patients were examined by light microscopy and immunohistochemistry (series 1), while specimens from another 17 patients were examined by light and transmission electron microscopy (series 2). Slight to marked angiogenesis was found in 5 patients from series 1 and 7 patients from series 2. Ultrastructural examination indicated that these blood vessels were more fragile than those in the normal eighth cranial nerve. These findings suggested that sudden hearing loss in vestibular schwannoma patients may be related to the disruption of such blood vessels. In addition, surgical injury to these blood vessels may cause postoperative hearing loss despite preservation of the cochlear nerve.

Nerve growth factor receptor expression in heterotransplanted vestibular schwannoma in athymic nude mice.

Nerve growth factor (NGF) has the potential to induce cellular differentiation in various neoplastic and non-neoplastic cell lines. The purpose of the present study was to determine by immunohistochemistry: the presence/distribution of nerve growth factor receptor (NGFr), cellular proliferation expressed by Ki-67, and intratumoral vascularization visualized by the endothelial marker CD 31, in a series of 61 human vestibular schwannoma heterotransplants in athymic nude mice. The immunohistochemical results were correlated to the observed macroscopic growth in 22 heterotransplants (36%) with obvious macroscopic growth, versus 39 heterotransplants (64%), that were stationary or regressed. The positive immunoreactivity to NGFr, number of Ki-67 positive nuclei and number of intratumoral vessels were significantly higher in the 22 (36%) growing heterotransplants than in the 39 heterotransplants (64%), which were stationary or regressed (p < 0.00005, p = 0.046, p < 0.00001). NGFr was statistically related to the vascularity of the heterotransplants expressed by CD 31 (p<0.00001). No significant relation was observed between NGFr and the proliferation, as estimated by Ki-67. The results revealed that the macroscopic growth of VS in athymic nude mice was associated with strong positive expression of NGFr, high cellular proliferation expressed by Ki-67 and vivid neovascularization expressed by CD 31. The possible clinical applicability of the achieved results is discussed.

Expression of steroid receptors in acoustic neuroma.

The expression of steroid receptors has been investigated in an attempt to clarify the role of steroid hormones in the proliferation and progression of acoustic neuromas. Specimens of tumours taken during translabyrinthine surgery were tested for cytosolic (c) and nuclear (n) steroid receptors. Oestrogen and progesterone receptor levels were evaluated by enzymatic immuno-assay, while androgen receptor binding levels were detected by dextran-coated charcoal method in a single-step determination. In some cases, the six point Scatchard analysis of cytosolic and nuclear androgen receptor was also performed. Threshold values were: 3 fmol/mg of proteins for cytosolic steroid receptors and 20 fmol/mg DNA for nuclear steroids, which corresponded to approximate median values of cytosolic and nuclear oestrogen and progesterone, respectively. Oestrogen and progesterone appeared to be localized more frequently in the nuclei rather than in the cytosol (70% oestrogen and progesterone positivity in the nuclei; 30% oestrogen, 40% progesterone positivity in the cytosol), while androgen receptors were preferentially localized in the cytosol (80% positivity in the cytosol; 40% positivity in the nuclei). A negative non-linear correlation between cytosolic oestrogen and cytosolic androgen receptors was found. There was a direct linear correlation between cytosolic oestrogen and nuclear oestrogen levels. A strict correlation between nuclear oestrogen and nuclear progesterone incidence was shown. Preliminary analysis of clinical data and biochemical parameters showed that cytosolic progesterone levels inversely correlated with tumour size.

Ultrastructure of the vestibular dark cell area in patients with acoustic neurinoma.

The human vestibular dark cell (DC) areas of the utricle and ampulla of the lateral semicircular canal were investigated ultrastructurally in 7 patients with acoustic neurinoma. Two types of DCs mainly constituted the epithelial cells of the DC area. One type of DC had basolateral infoldings that were closely interwoven with the melanocyte processes, and the pinocytotic vesicles were frequently found within the basolateral infoldings and the melanocyte processes. The other type of DC had basolateral infolsings stacked upon each other. The former type of DC was more frequently found than the latter type in the DC area adjacent to the sensory epithelium and in the mid portion of the DC area. However, the latter type of DC was more predominant in the DC area distant from the sensory epithelium. These findings suggest that the activity of DCs is modulated by the melanocytes and that the former type of DC has a more active role in ion and/or fluid transport than the latter type of DC. Thus, the DC area near the sensory epithelium might be more actively engaged in the production and regulation of vestibular endolymph than that distant from the sensory epithelium.

The limitations of hearing preservation in acoustic neuroma surgery: histological study of the interface between the eighth cranial nerve and the tumor.

The interface between the eighth cranial nerve and acoustic neuroma was investigated by light microscopy and immunohistochemistry in paraffin sections in 13 patients, and in Epon sections in 19 patients. The cochlear nerve was severely invaded by tumor cells in two of six specimens examined, moderately invaded in two specimens, and not invaded in two specimens. Gliosis was frequently found within the cochlear and vestibular nerve. Hemangioma-like tissue was occasionally found attached to the cochlear and vestibular nerve adjacent to the tumor. These findings were considered to be related to difficulty in hearing preservation in some patients although acoustic neuroma surgery was successful.

Ultrastructure of melanocytes in the dark cell area of human vestibular organs: functional implications of gap junctions, isolated cilia, and annulate lamellae.

BACKGROUND: It is known that melanocytes exist in almost all parts of the inner ear, such as the cochlear duct, stria vascularis, Reissner's membrane, modiolus, vestibular organs in the region surrounding the cristae and maculae, semicircular canals, and pars rugosa of the endolymphatic sac. But there have been few studies using human materials, because of the difficulty of obtaining materials. We attempted to investigate the detailed ultrastructure of melanocytes in the vestibular organs of human inner ear. METHODS: Eight surgical specimens obtained from patients with vestibular schwannoma were studied by light microscopy and electron microscopy. RESULTS: Melanocytes were found in the subepithelial layer of the dark cell area. Melanocytes had round or spindle-shaped nuclei and clear cytoplasm with brown pigment granules. Besides melanocytes, there were melanophages, fibroblasts, and small blood vessels. Through electron microscopy we found melanocytes with round-shaped melanosomes in various stages of pigmentation, well-developed Golgi apparatus and endoplasmic reticulum in the cytoplasm, and many cytoplasmic processes. Gap junctions were occasionally found between the cytoplasmic processes. And there were pinocytotic vesicles just under the limiting membrane of melanocytes, and intermediate filaments were abundant in the cytoplasm. Isolated cilia of melanocytes, annulate lamellae, and fusiform banded structures in the connective tissue area around melanocytes were found. CONCLUSIONS: Melanocytes in human vestibular organs actively synthesize melanosomes. Frequent findings of isolated cilia and fusiform banded structures and the incidental existence of annulate lamellae may be an indicator of this metabolically activated state of melanocytes. Moreover, monitoring environmental changes by isolated cilia, melanocytes in the human inner ear could act not only as one cell but also as a group to achieve their physiological functions by means of information transmission through gap junctions.

Vestibular sensory epithelia in patients with acoustic neurinoma.

The human vestibular sensory epithelia of the macula utriculi were examined ultrastructurally in 13 cases with acoustic neurinoma. The sensory epithelia were fairly well preserved, with several morphological changes such as lipofuscin granules, intraepithelial cysts, pyknosis, vacuoles, laminated structures, banded structures and degeneration of subepithelial myelinated nerves evident. The pathological significance of these findings was discussed. There were thickened irregular membrane formations in spaces between the nerve calyces and type I hair cells which might be related to acoustic neurinoma. Some type I hair cells were incompletely surrounded by nerve calyces. The neck regions of these cells received direct contact from the efferent nerve endings.

Light and electron microscopic study of vestibular sensory epithelia in 17 cases with acoustic neurinoma.

Vestibular sensory epithelia were studied histologically and ultrastructurally in 17 cases with acoustic neurinoma (AN). The superior vestibular nerve (SVN) near the fundus was also histologically studied in 5 of these 17 cases. Histologically, severe fibrotic change of the vestibular sensory epithelia was found in 1 case, and severe fibrotic change of the SVN was also found in this case. Intra-epithelial cysts were found at the edge of the utricular sensory epithelia in 2 cases. The cysts consisted of the transitional epithelium and were filled with the darkly stained substances. Ultrastructurally, abnormal accumulation of darkly stained masses within the nerve ending and abnormal accumulation of the fibrillar material below the normal basal lamina were frequently observed. These morphological changes described above were regarded as the pathological changes due to AN. In addition, the relationship among the histologic changes, ultrastructural changes and clinical data were fully investigated, and some histologic and ultrastructural changes were regarded as artifacts or age-related changes.

Skeinoid fibers: an ultrastructural marker of neurogenic spindle cell tumors.

The article describes novel stromal fibrillar aggregates found in three cases of neurogenic spindle cell tumor and eight cases of small intestinal stromal tumor. The aggregates were composed of tangles of curvilinear fluffy fibrils with a periodicity of from 41 to 48 nm with a staining pattern similar to that of collagen fibrils. The overall ultrastructural appearance simulated skeins of yarn, hence they are designated skeinoid fibers. No similar stromal fibers were found in more than 5000 other tumors studied. Their exclusive presence in neurogenic spindle cell tumors suggests the possibility that they are an ultrastructural marker for neurogenic spindle cell tumors and that the eight cases of small intestinal stromal tumors with skeinoid fibers may be of neurogenic origin.

Peripheral nerve sheath tumours: an ultrastructural study of 30 cases.

This report deals with the ultrastructural observations of 30 peripheral nerve sheath tumours [PNST], which include 25 schwannomas of acoustic nerve, one schwannoma of cauda equina, one neurofibroma from a case of Von Recklinghausen's disease, one pigmented neurofibroma of spinal nerve root and a malignant schwannoma of frontal region. Interdigitating slender cytoplasmic processes covered with a continuous layer of basal lamina constitute the single most important ultrastructural attribute of Schwann cells. Myelin formation was encountered in the cell processes of four out of 25 acoustic schwannomas. In four cases Microtubular arrays identical to that in an axon were seen in Schwann cells. These two observations require further support by additional cases of PNST studies by electron microscopy. The neurofibroma consisted only of Schwann cells and no ultrastructurally identifiable perineurial cells or fibroblasts were detected. The cells in the pigmented neurofibroma revealed submicroscopic features of both Schwann cell and melanocyte, indicating their common ancestry. A unique case of malignant schwannoma arising from frontal meninges is illustrated and it is emphasized that electron microscopy is mandatory for a correct histogenetic diagnosis of malignant tumours which occur at unexpected anatomical sites.

Ultrastructural evidence of a merocrine secretion in the human endolymphatic sac.

The results of a light and transmission electron microscopic analysis of an endolymphatic sac (ES) from a patient suffering from episodic vertigo, tinnitus, and hearing loss are presented. A biopsy of the intraosseous portion of the ES was obtained during a translabyrinthine approach to section the vestibular nerve in the internal acoustic meatus. The material consisted mainly of tubular epithelial structures filled with heavily stained material. Pathologically dilated and degranulated rough endoplasmic reticuli and disaggregation of polyribosomes with accumulation of solitary ribosomes in the cytosol and endoplasmic reticulum suggested a disturbed epithelial cell protein synthesis. Ultrastructural evidence of an increased merocrine secretion of glycoprotein conjugates into the ES was noted. This made it possible to analyze the presumed intracellular secretory pathways. An increased number of intraepithelial lymphocytes and monocytes was observed. Since the inner ear had been subjected to surgical intervention before the vestibular nerve section, no conclusions can be drawn as to whether the patient's symptoms were related to the disturbed protein metabolism and hypersecretion of glycoprotein conjugates into the ES. The findings support earlier experimental results that indicate that the ES has not only a resorptive function but also a secretory one.

Ultrastructural localization of nerve growth factor receptor in acoustic neurinoma.

Light and electron microscopic immunohistochemical studies on nerve growth factor (NGF) receptors in acoustic neurinoma cases were made using a monoclonal antibody to human NGF receptor. Immunoreactivity of the NGF receptor was found exclusively on the cytoplasmic membrane of schwannoma cells. Reaction product was highly concentrated at the interdigitated distal end of the cell processes, but it was discontinuous in the perinuclear area of the cell body. Basal lamina was located outside the immunoreacted cytoplasmic membrane of the cell body, but it was not observed at the tip of the interdigitated cell processes. These findings suggest that NGF receptor expression has some relationship with the formation of interdigitated schwannoma cell processes. Other possibilities for the functional significance of the schwannoma NGF receptor are also discussed.