Anneliese Schrott-Fischer

Role of class D L-type Ca2+ channels for cochlear morphology.

Voltage-gated Ca(2+) channels formed by subunits (class D Ca(2+) channels) tightly regulate neurotransmitter release from cochlear inner hair cells (IHCs) by controlling the majority of depolarisation-induced Ca(2+) entry. We have recently shown that the absence of these channels can cause deafness and degeneration of outer hair cells (OHCs) and IHCs in alpha1D-deficient mice (alpha1D(-/-)) (Platzer et al., 2000. Cell 102, 89-97). We investigated the time-dependent patterns of degeneration during postnatal development in the alpha1D(-/-) mouse cochlea using light and electron microscopy. At postnatal day 3 (P3), electron microscopy revealed no morphological aberrations in sensory cells, in afferent as well as in efferent nerve endings. But at P7 we observed a beginning degeneration of afferent nerve fibres by electron microscopy. By P15, we found a loss of OHCs in apical turns but electron microscopy revealed no ultrastructural changes in IHCs and efferent axons as compared to C57 black control animals (C57BL). We demonstrated by serial ultrathin sectioning of 15 days old alpha1D(-/-) mice that intact efferent nerve fibres formed direct contacts with IHCs as the degeneration of afferent nerve fibres progressed. We also saw a notable degeneration of spiral ganglion cells at P15. By 8 months, nearly all spiral ganglion and sensory cells of the organ of Corti were absent. Random ultrathin sectioning gave the impression that synaptic bodies abundant in wild-type animals were absent in nearly all alpha1D(-/-) mice investigated. We conclude that besides presumably reduced synaptic bodies the absence of class D L-type Ca(2+) channels does not prevent morphological development of the cochlea until P3 but may cause cochlear degeneration thereafter. The observed pattern of degeneration involves afferent nerve fibres (P7) followed by cell bodies in the spiral ganglion (P15), OHCs (P15) and IHCs (after P15).

Quantitative evaluation of cochlear neurons and computer-aided three-dimensional reconstruction of spiral ganglion cells in humans with a peripheral loss of nerve fibres

Quantitative data on human cochlear neuronal elements were collected from various regions in five patients with high-tone hearing loss due to presbycusis and in two patients with normal hearing. The number of nerve fibers was assessed in the spiral lamina and in the inner acoustic meatus together with counts of spiral ganglion cells. The results show that the number of neurons decreased peripherally, i.e., with increasing distance from the central nervous system in patients with high-tone hearing loss due to presbycusis. In two patients with normal hearing no significant difference in the number of neurons was found in the lamina spiralis as compared to the inner acoustic canal. Computer-aided 3-dimensional reconstruction of the human spiral ganglion displayed large bipolar neurons (type I cells), but also large ganglion cells with one missing axon. The results may indicate that a slow retrograde degeneration occurs from the periphery towards the spiral ganglion in presbycusis. Transmission electron microscopy analysis of freshly fixed human spiral ganglions displayed interneural connections. It is speculated whether a trophic supply from other neurons at the level of the spiral ganglion can prevent or delay further degeneration of the central axon.

Localization of ChAT-like immunoreactivity in the vestibular endorgans of the rat

In vertebrates acetylcholine (ACh) has been generally considered as a neurotransmitter of the vestibular efferent system. The precise localization and innervation of the cholinergic nerve endings in the vestibular sensory periphery is still unknown. We examined choline acetyltransferase (ChAT)-like immunoreactivity in all five endorgans of the rat vestibule with light and electron microscopy using a modified pre-embedding immunostaining technique. The results were: (1) ChAT-like immunoreactivity was widespread in all five endorgans of the vestibule and confined to the vesiculated efferent nerve endings. (2) Two types of ChAT-like immunostained nerve endings can be identified according to their size and innervation pattern: a large nerve ending and a small--middle size one. (3) Vestibular endorgans differ in their ChAT-like immunoreactivity: staining is dense in the macula of the utricule and the three ampullary cristae, but less so in the macula of the saccule. (4) We found also a regional difference of the ChAT-like immunostaining in ampullary crista. ChAT-like immunostained nerve endings were predominant in the periphery close to the semilunar plane, and less in density in the central area. These findings demonstrate that ACh is a major neurotransmitter in the vestibular efferent system.

Structural/audiometric correlations in a human inner ear with noise-induced hearing loss

A morphological analysis was performed on a human cochlea removed during skull base surgery. The patient experienced a noise-induced hearing loss following 30 years of mechanical exposure. The tissue was processed according to the block surface technique and the organ of Corti, osseous spiral lamina and spiral ganglion were analyzed at different levels. There was a circumscribed lesion approx. 10 mm from the round window extending to about 13 mm. At this site, the dominant pathological feature was the loss of outer hair cells that was comprehensive in the centermost area and partial in the peripheral region of the damage. The degradation of inner hair cells was less severe with signs of cell atrophy yet with limited loss. Outer pillar cells were often collapsed leading to deformation of the acoustic ridge. The Deiters cells were often present and physically interactive with remaining nerve fibers. In the reticular lamina, surgical manipulation and dissection resulted in tears which may be attributed to a reduction of intercellular strength between cells. In the damaged area, there was a 45% loss of myelinated nerve fibers measured at the osseous spiral lamina. Pathological changes could not be observed in the spiral ganglion with certainty although the type II cells innervating the outer hair cells were often difficult to discern.

Connexin 26 in human fetal development of the inner ear.

Specialized for intercellular communication, gap junctions have been theorized to provide a means (the epithelial and connective tissue gap junction systems) by which fluid and ions might be transported for maintenance of high levels of endolymphatic K+ [Kikuchi et al., 1994. Acta Otolaryngol. 114, 520-528] in the inner ear. A primary constituent of these gap junctions is connexin 26 (Cx26), a protein encoded by the gene GJB2 and found in both epithelial and connective tissue cells. It has been shown that a mutation in Cx26 accounts for 50% of patients with autosomal recessive nonsyndromic hearing loss. In the present study, we document the emergence and distribution features of Cx26 through various stages (weeks 11-31) of gestation in human, fetal cochleae. Comparative patterns of Cx26 distribution are also presented in the mature rat. The cochleae were fixed in 4% paraformaldehyde within 2 h post mortem. Immunohistochemical studies were performed using a rabbit polyclonal antibody raised against synthetic peptide and corresponding with amino acids 108-122. Specimens were mounted into paraffin sections. Results show that Cx26-like immunoreactivity is evident at a prenatal age of 11 weeks and maintains a high intensity of reactivity through 31 weeks of gestation. The appearance of this reactivity seemed to modulate in parallel with the onset of development and histological maturation as well as provide functional maintenance. In the human fetal cochlea, Cx26-like immunoreactivity distribution resembled adult patterns by fetal week 20. At the completion of morphological development by week 31, reactivity appeared to achieve an adult profile of distribution. Descriptions and discussion of Cx26 distribution patterns are presented in detail.

Immunocytochemical detection of choline acetyltransferase in the human organ of Corti

In the mammalian cochlea acetylcholine has been considered a major neurotransmitter of the lateral and medial efferent fibers. The aims of the present study were to investigate the expression of ChAT in the human cochlea and to develop a new method for immunohistochemical investigations in the human cochlea both at the light and electronmicroscopic level. We thus examined the ChAT-like immunoreactivity in the human inner ear using light and electron microscopy with a pre-embedding technique. Our present results agree with the previously published data acquired in rodent species. The ChAT-like immunostaining could be found in the inner spiral fibers, the inner spiral bundle, tunnel crossing fibers and at the base of the outer hair cells. No staining was noted in the negative controls experiments, while rat cochleas used as positive controls showed the usual ChAT-like immunostaining as described above. The main difference between human and rat cochleas was that the efferent nerve supply seems to be less pronounced in the human cochleas.

Selective aspects of human pathology in high-tone hearing loss of the aging inner ear

Accompanied with aging, the thresholds for high frequency sounds may elevate and result in a progressive hearing loss described as presbycusis. Based on correlations between audiometric measures of aged patients and histologic findings garnered from postmortem examinations, four types of presbycusis have been characterized: sensory-neural, neural, strial, and conductive [Schuknecht, H.F., Gacek, M.R., 1993. Ann. Otol. Rhinol. Laryngol. 102, 1--16]. Otopathologic changes to the inner ear as a direct function of age, however, remain controversial. The focus of this investigation involves the pathological impact on remaining sensory structures in patients having sensory--neural degeneration. The current study presents seven human temporal bones extracted from patients aged 53--67 years with high-tone hearing loss and with no known history of extraordinary environmental events involving head or noise trauma, acoustic overstimulation, or ototoxicity. In previously published findings of these specimens, all but one temporal bone failed to demonstrate a meaningful correlation between audiometric measurements and loss of functional hair cell populations with secondary retrograde degeneration of nerve fibers. Using the block surface method, electron microscopic micrographs demonstrate ultrastructural changes in the cuticular plate, stereocilia, pillar cells, stria vascularis, and the spiral ligament. In all pathological specimens, the greatest incidence of degeneration was seen at the cuticular plate. Conclusively, our findings present three implications in the aging human cochlea: firstly, audiometric measures that represent a high-tone hearing loss may take various forms with respect to ultrastructural patterns of degeneration and surviving structures; secondly, the incidence of lipofuscin and lysosome granules does not correlate with the degree of hearing loss and; thirdly, as shown only in guinea pigs [Anniko, M., 1988. Scanning Microsc. 2, 1035--1041], high-tone hearing loss can be associated with deformation of the cuticular plate.

Ciliary Beat Frequency, Olfaction and Endoscopic Sinus Surgery

We assessed prospectively changes in olfaction and ciliary beat frequency (CBF) after functional endoscopic sinus surgery (FESS) in 70 patients with chronic sinusitis. CBF was measured by the microscopic-photometry technique 2, 4 and 6 months after FESS. Olfaction was evaluated by the Erlangener Smell Identification Test. Preoperative CBF was markedly reduced due to the infectious process. CBF was significantly improved (p < 0.001), reaching normal values 6 months postoperatively despite the endoscopic examination revealing normal nasal mucosa at around 3 months. The pattern of improvement of CBF was linear being more rapid between months 4 and 6. After operation olfactory-impaired patients were improved subjectively and objectively (p < 0.001 and p = 0.003). We conclude that impaired olfactory ability and CBF are significantly improved after FESS, and FESS demands longer postoperative follow-up periods for at least 6 months even if the clinical evaluations were normal.

Ultrastructural evaluation of calcitonin gene-related peptide immunoreactivity in the human cochlea and vestibular endorgans

Calcitonin gene‐related peptide (CGRP) is a neuropeptide widely distributed in the peripheral and central nervous system. Demonstrated in the efferent systems of the mammalian cochlea and vestibule, immunoreactive patterns of CGRP may vary by species. There is, however, no information in the literature investigating CGRP localization in the human cochlea. In the present study, the ultrastructural localization of CGRP immunoreactivity was evaluated in the human inner ear with immunoelectron microscopy. It was found that, in human cochlea, CGRP immunoreactivity was located in unmyelinated nerve fibres of the spiral lamina, inner spiral fibres beneath inner hair cells, tunnel spiral fibres, tunnel crossing fibres and outer radial fibres. In endorgans of human vestibule, CGRP immunoreactivity was located in vesiculated nerve fibres and bouton‐type nerve terminals which were seen to contact afferent nerve chalices surrounding type I sensory cells and afferent nerve fibres, or to form an en passant contact with afferent dendrites. CGRP immunoreactivity appeared to be confined to efferent systems in all cases. This study presents evidence that CGRP could serve a role in neurotransmission or neuroregulation in both cochlear and vestibular efferent systems of human.

Co-distribution patterns of chromogranin b-like immunoreactivity with chromogranin a and secretoneurin within the human brainstem

As members of the chromogranin family, chromogranin A, chromogranin B, and secretogranin II are acidic proteins found in large, dense core vesicles. They are endoproteolytically processed to smaller peptides and released after neuronal stimulation. Using immunocytochemistry, this study closely examines chromogranin B-like immunoreactivity within the human brainstem and then takes a comparative view of co-distribution patterns by chromogranin B, chromogranin A, and secretogranin II. We used an antiserum raised against a synthetic peptide (PE-11) present in the chromogranin B molecule. Secretogranin II was localized with an antiserum against secretoneurin, a 33 amino acid peptide, found within the secretogranin II precursor. Like chromogranin A and secretoneurin, chromogranin B is expressed through all levels of the human brainstem. Chromogranin B was exclusively detected in neuronal structures. The medial part of the substantia nigra pars reticulata, the nucleus interpeduncularis, the area of the central gray, and the raphe complex displayed a high density of PE-11-like immunoreactivity. Furthermore, a prominent staining was found in the medial, dorsal and gelatinous subnuclei of the solitary tract and the dorsal motor nucleus of vagus. The substantia gelatinosa of the caudal trigeminal nucleus and spinal cord were also very strongly PE-11-immunopositive. In conclusion, chromogranin B and secretogranin II showed similar distributions while neuronal localization typically differed from chromogranin A aside from a few exceptions. These findings may provide a framework for future research in revealing a functional role of chromogranin peptides in the human brainstem.