| 1. |
- Anderson, Malin, et al.
(författare)
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Structure and locomotion of adult in vitro regenerated spiral ganglion growth cones : a study using video microscopy and SEM
- 2006
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Ingår i: Hearing Research. - : Elsevier BV. - 0378-5955 .- 1878-5891. ; 215:1-2, s. 97-107
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Boström, Marja, et al.
(författare)
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Neural network and "Ganglion" formations in vitro : a video microscopy and scanning electron microscopy study on adult cultured spiral ganglion cells.
- 2007
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Ingår i: Otology and Neurotology. - 1531-7129 .- 1537-4505. ; 28:8, s. 1109-1119
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Tidskriftsartikel (refereegranskat)abstract
- 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 beta-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.
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| 3. |
- Hayashi, Hisamitsu, et al.
(författare)
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Molecular organization and fine structure of the human tectorial membrane : is it replenished?
- 2015
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 362:3, s. 513-527
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Tidskriftsartikel (refereegranskat)abstract
- Auditory sensitivity and frequency resolution depend on the physical properties of the basilar membrane in combination with outer hair cell-based amplification in the cochlea. The physiological role of the tectorial membrane (TM) in hair cell transduction has been controversial for decades. New insights into the TM structure and function have been gained from studies of targeted gene disruption. Several missense mutations in genes regulating the human TM structure have been described with phenotypic expressions. Here, we portray the remarkable gradient structure and molecular organization of the human TM. Ultrastructural analysis and confocal immunohistochemistry were performed in freshly fixed human cochleae obtained during surgery. Based on these findings and recent literature, we discuss the role of human TMs in hair cell activation. Moreover, the outcome proposes that the α-tectorin-positive amorphous layer of the human TM is replenished and partly undergoes regeneration during life.
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| 4. |
- Liu, Wei, et al.
(författare)
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Distribution of P75 neurotrophin receptor in adult human cochlea : an immunohistochemical study
- 2012
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 348:3, s. 407-415
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Tidskriftsartikel (refereegranskat)abstract
- Mechanisms underlying the unique survival property of human spiral neurons are yet to be explored. P75 (p75(NTR)) is a low affinity receptor for neurotrophins and is known to interact with Trk receptors to modulate ligand binding and signaling. Up-regulation of this receptor was found to be associated with apoptosis as well as with cell proliferation. Its distribution and injury-induced change in expression pattern in the cochlea have been mainly studied in rodents. There is still no report concerning p75(NTR) in post-natal human inner ear. We analyzed, for the first time, p75(NTR) expression in five freshly fixed human cochleae by using immunohistochemistry techniques, including myelin basic protein (MBP) as a myelin sheath marker and TrkB as the human spiral neuron marker, and by using thin optical sectioning of laser confocal microscopy. The inner ear specimens were obtained from adult patients who had normal pure tone thresholds before the surgical procedures, via a trans-cochlear approach for removal of giant posterior cranial fossa meningioma. The expression of p75(NTR) was investigated and localized in the glial cells, including Schwann cells and satellite glial cells in the Rosenthal canal, in the central nerve bundles within the modiolus, and in the osseous spiral lamina of the human cochleae. The biological significance of p75(NTR) in human cochlea is discussed.
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| 5. |
- Liu, Wei, et al.
(författare)
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Expression of trans-membrane serine protease 3 (TMPRSS3) in the human organ of Corti
- 2018
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 372:3, s. 445-456
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Tidskriftsartikel (refereegranskat)abstract
- TMPRSS3 (Trans-membrane Serine Protease 3) is a type II trans-membrane serine protease that has proteolytic activity essential for hearing. Mutations in the gene cause non-syndromic autosomal recessive deafness (DFNB8/10) in humans. Knowledge about its cellular distribution in the human inner ear may increase our understanding of its physiological role and involvement in deafness, ultimately leading to therapeutic interventions. In this study, we used super-resolution structured illumination microscopy for the first time together with transmission electron microscopy to localize the TMPRSS3 protein in the human organ of Corti. Archival human cochleae were dissected out during petroclival meningioma surgery. Microscopy with Zeiss LSM710 microscope achieved a lateral resolution of approximately 80 nm. TMPRSS3 was found to be associated with actin in both inner and outer hair cells. TMPRSS3 was located in cell surface-associated cytoskeletal bodies (surfoskelosomes) in inner and outer pillar cells and Deiters cells and in subcuticular organelles in outer hair cells. Our results suggest that TMPRSS3 proteolysis is linked to hair cell sterociliary mechanics and to the actin/microtubule networks that support cell motility and integrity.
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| 6. |
- Liu, Wei, et al.
(författare)
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Human cochlear microanatomy - an electron microscopy and super-resolution structured illumination study and review
- 2020
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Ingår i: HEARING BALANCE AND COMMUNICATION. - : ROUTLEDGE JOURNALS, TAYLOR & FRANCIS LTD. - 2169-5717 .- 2169-5725. ; 18:4, s. 256-269
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Forskningsöversikt (refereegranskat)abstract
- Objective: Studies of the human cochlea are particularly challenging due its exceptional vulnerability and surrounding hard bone. Swift fixation and mild decalcification are necessary to maintain its structural integrity and preserve antigenicity. Such procedures may allow immunohistochemistry, gene analyses, and molecular imaging using super resolution illumination microscopy (SR-SIM) with nanometer resolution. Design: This presentation updates recent studies of the human cochlear microanatomy and immunohistochemistry by our laboratory, discussed in the context of current and past anatomic findings and the available literature. Results: Human studies are necessary, and there are intriguing discrepancies compared with experimental animal studies, highlighting that "men are not simply big mice." The results may improve our understanding of the function of the human hearing organ, the diseases related to it, and how this better understanding can be extended to impact future treatment. Conclusion: The first human inner ear gene therapy trials are in progress, and the accessibility of human cochlear tissue for future stem cell treatment and gene transfer needs further elucidation.
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| 7. |
- Liu, Wei, et al.
(författare)
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Macromolecular organization and fine structure of the human basilar membrane - RELEVANCE for cochlear implantation
- 2015
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 360:2, s. 245-262
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Tidskriftsartikel (refereegranskat)abstract
- Introduction Cochlear micromechanics and frequency tuning depend on the macromolecular organization of the basilar membrane (BM), which is still unclear in man. Novel techniques in cochlear implantation (CI) motivate further analyses of the BM. Materials and methods Normal cochleae from patients undergoing removal of life-threatening petro-clival meningioma and an autopsy specimen from a normal human were used. Laser-confocal microscopy, high resolution scanning (SEM) and transmission electronmicroscopy (TEM) were carried out in combination. In addition, one human temporal bone was decellularized and investigated by SEM. Results The human BM consisted in four separate layers: (1) epithelial basement membrane positive for laminin-beta 2 andcollagen IV, (2) BM Bproper boolean AND composed of radial fibers expressing collagen II and XI, (3) layer of collagen IV and (4) tympanic covering layer (TCL) expressing collagen IV, fibronectin and integrin. BM thickness varied both radially and longitudinally (mean 0.55-1.16 mu m). BM was thinnest near the OHC region and laterally. Conclusions There are several important similarities and differences between the morphology of the BM in humans and animals. Unlike in animals, it does not contain a distinct pars tecta (arcuate) and pectinata. Its width increases and thickness decreases as it travels apically in the cochlea. Findings show that the human BM is thinnest and probably most vibration-sensitive at the outer pillar feet/Deiter cells at the OHCs. The inner pillar and IHCs seem situated on a fairly rigid part of the BM. The gradient design of the BM suggests that its vulnerability increases apical wards when performing hearing preservation CI surgery.
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| 8. |
- Liu, Wei, et al.
(författare)
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Molecular composition and distribution of gap junctions in the sensory epithelium of the human cochlea a super-resolution structured illumination microscopy (SR-SIM) study
- 2017
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 122:3, s. 160-170
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Tidskriftsartikel (refereegranskat)abstract
- Background: Mutations in the GJB2 gene, which encodes the Connexin26 (Cx26) protein, are the most common cause of childhood hearing loss in American and European populations. The cochlea contains a gap junction (GJ) network in the sensory epithelium and two connective tissue networks in the lateral wall and spiral limbus. The syncytia contain the GJ proteins beta 2 (GJB2/Cx26) and beta 6 (GJB6/Cx30). Our knowledge of their expression in humans is insufficient due to the limited availability of tissue. Here, we sought to establish the molecular arrangement of GJs in the epithelial network of the human cochlea using surgically obtained samples. Methods: We analyzed Cx26 and Cx30 expression in GJ networks in well-preserved adult human auditory sensory epithelium using confocal, electron, and super -resolution structured illumination microscopy (SR-SIM). Results: Cx30 plaques (<5 mu m) dominated, while Cx26 plaques were subtle and appeared as 'mini junctions' (2-300 nm). 3-D volume rendering of Z-stacks and orthogonal projections from single optical sections suggested that the GJs are homomeric/homotypic and consist of assemblies of identical GJs composed of either Cx26 or Cx30. Occasionally, the two protein types were co-expressed, suggesting functional cooperation. Conclusions: Establishing the molecular composition and distribution of the GJ networks in the human cochlea may increase our understanding of the pathophysiology of Cx-related hearing loss. This information may also assist in developing future strategies to treat genetic hearing loss.
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| 9. |
- Liu, Wei, et al.
(författare)
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Possible role of gap junction intercellular channels and connexin 43 in satellite glial cells (SGCs) for preservation of human spiral ganglion neurons : A comparative study with clinical implications
- 2014
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 355:2, s. 267-278
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Tidskriftsartikel (refereegranskat)abstract
- Human spiral ganglion (SG) neurons show remarkable survival properties and maintain electric excitability for a long time after complete deafness and even separation from the organ of Corti, features essential for cochlear implantation. Here, we analyze and compare the localization and distribution of gap junction (GJ) intercellular channels and connexin 43 (Cx43) in cells surrounding SG cell bodies in man and guinea pig by using transmission electron microscopy and confocal immunohistochemistry. GJs and Cx43 expression has been recognized in satellite glial cells (SGCs) in non-myelinating sensory ganglia including the human SG. In man, SG neurons can survive as mono-polar or "amputated" cells with unbroken central projections following dendrite degeneration and consolidation of the dendrite pole. Cx43-mediated GJ signaling between SGCs is believed to play a key role in this "healing" process and could explain the unique preservation of human SG neurons and the persistence of cochlear implant function.
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| 10. |
- Liu, Wei, et al.
(författare)
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The Human "Cochlear Battery" - Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea
- 2017
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Ingår i: Frontiers in Molecular Neuroscience. - : Frontiers Media SA. - 1662-5099. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: The cochlea produces an electric field potential essential for hair cell transduction and hearing. This biological "battery" is situated in the lateral wall of the cochlea and contains molecular machinery that secretes and recycles K+ ions. Its functioning depends on junctional proteins that restrict the para-cellular escape of ions. The tight junction protein Claudin-11 has been found to be one of the major constituents of this barrier that maintains ion gradients (Gow et al., 2004; Kitajiri et al., 2004a). We are the first to elucidate the human Claudin-11 framework and the associated ion transport machinery using super-resolution fluorescence illumination microscopy (SR-SIM). Methods: Archival cochleae obtained during meningioma surgery were used for SR-SIM together with transmission electron microscopy after ethical consent. Results: Claudin-11-expressing cells formed parallel tight junction lamellae that insulated the epithelial syncytium of the stria vascularis and extended to the suprastrial region. Intercellular gap junctions were found between the barrier cells and fibrocytes. Conclusion: Transmission electron microscopy, confocal microscopy and SR-SIM revealed exclusive cell specialization in the various subdomains of the lateral wall of the human cochlea. The Claudin-11-expressing cells exhibited both conductor and isolator characteristics, and these micro-porous separators may selectively mediate the movement of charged units to the intrastrial space in a manner that is analogous to a conventional electrochemical "battery." The function and relevance of this battery for the development of inner ear disease are discussed.
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