Marja Boström

Unique expression of connexins in the human cochlea

Mutations in the genes GJB2 and GJB6, which encode the proteins Connexin 26 (Cx26) and Connexin 30 (Cx30), have been linked to nonsyndromic prelingual deafness in humans. These proteins may form so-called gap junctions (GJ) or transcellular pathways between cells. The pathogenesis of deafness due to GJ Connexin mutations remains unclear partly because examinations performed in the human ear are infrequent. Here we analysed the expression and distribution of Cx26 and Cx30 in five fresh normal human cochleae taken out at occasional surgery. Immunohistochemistry including confocal microscopy in decalcified specimen showed that these proteins are widely expressed in the human cochlea. In the lateral wall there was strong antibody co-labeling for Cx26 and Cx30 that support the existence of channels comprising heteromeric Cx26/Cx30 connexons. In the organ of Corti there were some co-labeling in the supporting cell area including mainly the Claudius cells and Deiter cells of these two Cxs, apart from isolated Cx26 and Cx30 labeling in the same area, suggestive of both homomeric/homotypic pattern and hybrid pattern (heteromeric or heterotypic). Cx30, Cx26 and Connexin 36 (Cx36) immunoreactivity was also associated with spiral ganglion type I neurons, the latter being a gap junction protein specific to neurons. Gap-junction-based electrical synapses are not known to occur in mammalian auditory system other than in bats where they may play a role for fast electrical nerve transmission useful for echolocation. Their potential role in the processing of human auditory nerve signaling as well as non-GJ roles of the connexins in human cochlea is discussed.

Expression of peripherin in human cochlea

The organ of Corti contains two different types of auditory receptors; the inner (IHCs) and outer (OHCs) hair cells. This dualism is further represented in their innervation, IHCs being innervated by type I neurons, and OHCs by type II neurons (in man, named small ganglion cells). Two efferent systems are also present. Here, we have analyzed the expression of the 57-kDa neuron-specific intermediate filament protein peripherin (PP) in human cochlea. In the human organ of Corti, PP seems to be specifically expressed in OHC afferents. Small or type II spiral ganglion cell bodies also intensely express PP. Thus, PP can be used as a marker for the characterization of the innervation of the OHC system in man.

Effects of Neurotrophic Factors on Growth and Glial Cell Alignment of Cultured Adult Spiral Ganglion Cells

Adult spiral ganglion cells were cultured in chorus to assess the influence of the neurotrophins brain-derived neurotrophic factor, neurotrophin 3 and glial cell line-derived neurotrophic factor (GDNF) on neurite growth and Schwann cell alignment. Over 1500 measurements were collected using each factor at 10 ng/ml and all three in combination. Evaluation was made with GDNF at concentrations of up to 100 ng/ml. Neurite dimensions were assessed at days 5, 7, 9 and 11 using a computer-based program (Axon Analyzer). GDNF had a strong effect on spiral ganglion cell growth almost attaining the level of all three factors in combination. GDNF increased glial cell alignment and nerve bundle formation. Results show the potential of GDNF to maintain and possibly restore auditory nerve integrity.

Expression of peripherin in the pig spiral ganglion – aspects of nerve injury and regeneration

Conclusion. Peripherin protein may be important not only for developing neurons but also for the maintenance and regeneration of axonal processes in the mature cochlea. More knowledge about its expression and function could improve our understanding with reference to axonal regrowth and nerve restoration in the damaged cochlea. Objective. To investigate the existence of peripherin protein in adult pig spiral ganglion and cultured spiral ganglion neurons of the guinea pig. Materials and methods. Immunohistochemistry with anti-peripherin antibodies was performed on sections of adult pig spiral ganglion and guinea pig spiral ganglion cell (SGC) culture. Results. In pig auditory neurons, both type I and type II SGCs showed expression of the protein peripherin. These cells were not preferentially located near the intraganglionic spiral bundle (IGSB). The IGSB consisted of thin calibre fibres showing intense peripherin and thicker fibres that were TUJ-1 positive. Only a few fibres within the IGSB co-expressed both peripherin and TUJ-1. Cultured guinea pig neurons displayed a rich expression of peripherin, including the nuclei. This protein was expressed in regions such as the perikaryon and axons but there was also a segmental expression of peripherin in some regions. Peripherin was more expressed in areas of axon branching and in the centre of the active growth cones and lammelipodia.

Structure and locomotion of adult in vitro regenerated spiral ganglion growth cones - A study using video microscopy and SEM

Neuronal development and neurite regeneration depends on the locomotion and navigation of nerve growth cones (GCs). There are few detailed descriptions of the GC function and structure in the adult auditory system. In this study, GCs of adult dissociated and cultured spiral ganglion (SG) neurons were analyzed in vitro utilizing combined high resolution scanning electron microscopy (SEM) and time lapse video microscopy (TLVM). Axon kinesis was assessed on planar substratum with growth factors BDNF, NT-3 and GDNF. At the nano-scale level, lamellipodial abdomen of the expanding GC was found to be decorated with short surface specializations, which at TLVM were considered to be related to their crawling capacity. Filopodia were devoid of these surface structures, supporting its generally described sensory role. Microspikes appearing on lamellipodia and axons, showed circular adhesions, which at TLVM were found to provide anchorage of the navigating and turning axon. Neurons and GCs expressed the DCC-receptor for the guidance molecule netrin-1. Asymmetric ligand-based stimulation initiated turning responses suggest that this attractant cue influences steering of GC in adult regenerating auditory neurites. Hopefully, these findings may be used for ensuing tentative navigation of spiral ganglion neurons to induce regenerative processes in the human ear.

Expression of TrkB and BDNF in human cochlea—an immunohistochemical study

Surgical human cochlear specimens were obtained during the removal of large posterior cranial fossa meningioma by a transcochlear approach in which the cochlea was removed for maximal exposure of the tumor and protection of important structures, such as the brainstem, cranial nerves, and pivotal blood vessels. The cochlear tissue was fixed and cryo-sectioned for tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. TrkB receptor protein was expressed in both neuronal somata and the processes of human spiral ganglion neurons (SGNs). In the human organ of Corti, TrkB immunoreactivity was mainly present in nerve fibers underneath outer hair cells. BDNF expression was found neither in the organ of Corti nor in the spiral ganglion of human cochlea. For antibody specificity and for control and comparative purposes, TrkB immunocytochemistry was performed in primary cultures of cochlear neuron/glia from adult guinea pig. Confocal laser scanning microscopy showed that TrkB was homogeneously distributed in the cytoplasm of both neuronal somata and axons. Knowledge of the expression of TrkB receptor in human cochlea should help to determine the target structures for neuron preservation in hearing-impaired patients. Our results indicate that the regeneration of SGNs under pathological conditions can be enhanced with BDNF/TrkB-based pharmaceutical or genetic strategies.

Neural network and "ganglion" formations in vitro: a video microscopy and scanning electron microscopy study on adult cultured spiral ganglion cells.

Hypothesis: To analyze if adult-dissociated spiral ganglion cells may be propagated in vitro for later use in transplantation models to form integrated neural networks. Background: Hearing loss is often associated with primary or secondary spiral ganglion cell degeneration. New strategies for cell repair and tissue engineering warrants further elucidation of the regenerative capacity of the auditory nerve. Methods: We used in vitro/in video microscopy in combination with immunocytochemistry and field emission scanning electron microscopy to analyze neural development and network formation from dissociated adult guinea pig spiral ganglion cells. Cells were cultured in serum-free medium and in the presence of brain-derived neurotrophic factor, neurotrophin 3, and glia cell line-derived neurotrophic factor for up to 8 weeks. Results: Time-lapse video microscopy and scanning electron microscopy exposed the propagation of auditory neurons and the role of neural growth cones in axon locomotion, fasciculation, and nuclear migration, often ensuing in cell congregation (ganglion-like formations) during network formation. Axons were sometimes ensheathed by adjoining S-100/glia fibrillary acidic protein-expressing cells. A few expanding neurons were nestin positive and sometimes incorporated the markers of proliferating cells Ki67 and 5′-bromo-2-deoxyuridine. Neurons expressed the markers and transcription factors for neural development neurogenin 1, neurogenic differentiation factor 1, Brn3a, and GATA binding protein 3, as well as the neural markers β-III tubulin, NeuN, and neurofilament 160 during this process. Conclusion: This method of culturing and expanding spiral ganglion neurons in vitro may be useful in further studies of cell transplantation models aiming to restore the injured inner ear.

Immunolocalization of prestin in the human cochlea

Abstract Objective: Prestin is an anion-transporter-related protein highly expressed in mammalian outer hair cells (OHCs). It is associated with the OHC basolateral plasma membrane and responsible for cell body contraction, increasing cochlear sensitivity and frequency resolution. Here, we analysed the expression and distribution of prestin in the human cochlea. Study Design: Immunohistochemistry including confocal microscopy and SEM were performed on EDTA-decalcified human cochlea removed during petro-clival meningioma surgery. Results: Prestin was found to be expressed solely in OHCs. No staining was seen in IHCs. Prestin immunolabelling framed the OHCs along the sensory region of the human cochlea. Staining was most prominent in the lateral cell membrane with less expression in the sub- and peri-nuclear part of the cell. Prestin immunostaining was also detected in the OHC cytosol, presumably reflecting intracellular molecular trafficking. Conclusion: This study describes for the first time the localization and distribution of prestin in the well preserved human organ of Corti.

In vitro growth of Human endolymphatic sac cells: A transmission electron microscopic and immunohistochemical study in patients with vestibular schwannoma and Ménière's disease

Hypothesis Human endolymphatic sac cells have been notoriously difficult to maintain in culture. It was hypothesized that an in vitro environment intended for growth of keratinocytes would also be suitable for human endolymph sac cells. Background Studies on cell physiology of human endolymphatic sac cells have been hampered by difficulties in maintaining them in culture. Methods Human endolymphatic sac cells were taken from 10 patients during translabyrinthine skull base surgery for vestibular schwannoma, one of whom also had Ménières disease. Cell lines of proliferating epithelial cells were obtained after trypsinization and growth in a 3:1 mixture of Dulbeccos modified Eagle medium and Hams F12 medium supplemented with 10% fetal calf serum. Fibroblast overgrowth was counteracted by the use of so-called cloning rings. During various stages, cells were investigated with transmission electron microscopy and/or immunohistochemistry. Results Proliferation took place after 2 to 3 days of primary cell culture. The cells were cytokeratin-positive and pleomorphic, and they had abundant polarized microvillus-like projections, numerous coated cytoplasmic pits and vesicles, and a well-developed rough endoplasmic reticulum. Conclusion Cell lines of proliferating human endolymphatic sac cells can be produced with the technique described here and may be a valid tool in studies of human endolymph sac physiology.

Differentiation of human neural progenitor cell-derived spiral ganglion-like neurons : a time-lapse video study

Abstract Conclusions: Human neural progenitor cells can differentiate into spiral ganglion-like cells when exposed to inner ear-associated growth factors. The phenotype bears resemblance to human sphere-derived neurons. Objective: To establish an in vitro model for the human auditory nerve to replace and complement in vivo animal experiments and ultimately human in vivo transplantation. Methods: Human neural progenitors were differentiated under conditions developed for in vitro survival of human primary spiral ganglion culture with media containing growth factors associated with inner ear development. Differentiation was documented using time-lapse video microscopy. Time-dependent marker expression was evaluated using immunocytochemistry with fluorescence and laser confocal microscopy. Results: Within 14 days of differentiation, neural progenitors adopted neural phenotype and expressed spiral ganglion-associated markers.