Fredrik Edin

The Pre- and Post-Somatic Segments of the Human Type I Spiral Ganglion Neurons - Structural and Functional Considerations Related to Cochlear Implantation

Highlights • Pre- and post-somatic segments of type I spiral ganglion neurons (SGNs) are unmyelinated in man.• Following hair cell loss and retrograde nerve degeneration SGNs survive as “mono-polar” cells in human deafness.• Non-myelinated Schwann cells may consolidate the neural cell bodies and protect SGNs from further degeneration.• Human SGNs can persist as electrically excitable mono-polar cells even after long-time deafness.• Robust survival of human SGNs is a prerequisite for cochlear implant function.

Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

Ischaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man.

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

Molecular composition and distribution of gap junctions in the sensory epithelium of the human cochlea a super-resolution structured illumination microscopy (SR-SIM) study

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 μm) 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.

3-D gel culture and time-lapse video microscopy of the human vestibular nerve

Abstract Conclusions: Human inner ear neurons have an innate regenerative capacity and can be cultured in vitro in a 3-D gel. The culture technique is valuable for experimental investigations of human inner ear neuron signaling and regeneration. Objectives: To establish a new in vitro model to study human inner ear nerve signaling and regeneration. Methods: Human superior vestibular ganglion (SVG) was harvested during translabyrinthine surgery for removal of vestibular schwannoma. After dissection tissue explants were embedded and cultured in a laminin-based 3-D matrix (Matrigel™). 3-D growth cone (GC) expansion was analyzed using time-lapse video microscopy (TLVM). Neural marker expression was appraised using immunocytochemistry with fluorescence and laser confocal microscopy. Results: Tissue explants from adult human SVG could be cultured in 3-D in a gel, indicating an innate potential for regeneration. Cultured GCs were found to expand dynamically in the gel. Growth cone expansion and axonal Schwann cell alignment were documented using TLVM. Neurons were identified morphologically and through immunohistochemical staining.

NANOCI—Nanotechnology Based Cochlear Implant With Gapless Interface to Auditory Neurons

Cochlear implants (CI) restore functional hearing in the majority of deaf patients. Despite the tremendous success of these devices, some limitations remain. The bottleneck for optimal electrical stimulation with CI is caused by the anatomical gap between the electrode array and the auditory neurons in the inner ear. As a consequence, current devices are limited through 1) low frequency resolution, hence sub-optimal sound quality and 2), large stimulation currents, hence high energy consumption (responsible for significant battery costs and for impeding the development of fully implantable systems). A recently completed, multinational and interdisciplinary project called NANOCI aimed at overcoming current limitations by creating a gapless interface between auditory nerve fibers and the cochlear implant electrode array. This ambitious goal was achieved in vivo by neurotrophin-induced attraction of neurites through an intracochlear gel-nanomatrix onto a modified nanoCI electrode array located in the scala tympani of deafened guinea pigs. Functionally, the gapless interface led to lower stimulation thresholds and a larger dynamic range in vivo, and to reduced stimulation energy requirement (up to fivefold) in an in vitro model using auditory neurons cultured on multi-electrode arrays. In conclusion, the NANOCI project yielded proof of concept that a gapless interface between auditory neurons and cochlear implant electrode arrays is feasible. These findings may be of relevance for the development of future CI systems with better sound quality and performance and lower energy consumption. The present overview/review paper summarizes the NANOCI project history and highlights achievements of the individual work packages.

Distribution of pejvakin in human spiral ganglion: An immunohistochemical study

Abstract Up to 10% of permanent hearing impairments in children originate from lesions in the neuronal auditory pathway. This form of auditory neuron injury called auditory neuropathy features a preservation of outer hair cell integrity but an impaired inner hair cell function and/or neuronal transmission. DFNB59 gene encodes the protein pejvakin (PJVK) and its mutations cause autosomal recessive auditory neuropathy as well as other forms of sensorineural hearing loss. The finding of distinct forms of hearing anomalies was based on studies of consanguineous families from different ethnic groups as well as studies in mice with PJVK gene mutations. In the present immunohistochemical study, the distribution of pejvakin protein in surgically obtained human cochleae was for the first time investigated. The human cochleae had normal hearing thresholds before the operation. The expression of pejvakin was located in the cell bodies of all spiral ganglion neurons rather than the nerve fibers that were labeled with Tuj 1 antibody. As Tuj 1 antibody stained the cytoplasm of Type 1 cells, pejvakin antibody labeled both type 1 and type 2 cells. The nuclei of the neurons were also PJVK-positive. No labeling was seen in the structures within the organ of Corti and the stria vascularis. In the previous study, PJVK had been detected in the hair cells, the spiral ganglion, the cochlear nuclei, the superior olivary nucleus, and the inferior colliculus in mouse. Our study demonstrated for the first time the expression of PJVK in human spiral ganglion neurons. Its functional role in neural signal propagation and synchrony needs further elucidation.

The effect of pulsed electric fields on the electrotactic migration of human neural progenitor cells through the involvement of intracellular calcium signaling

Endogenous electric fields (EFs) are required for the physiological control of the central nervous system development. Application of the direct current EFs to neural stem cells has been studied for the possibility of stem cell transplantation as one of the therapies for brain injury. EFs generated within the nervous system are often associated with action potentials and synaptic activity, apparently resulting in a pulsed current in nature. The aim of this study is to investigate the effect of pulsed EF, which can reduce the cytotoxicity, on the migration of human neural progenitor cells (hNPCs). We applied the mono-directional pulsed EF with a strength of 250mV/mm to hNPCs for 6h. The migration distance of the hNPCs exposed to pulsed EF was significantly greater compared with the control not exposed to the EF. Pulsed EFs, however, had less of an effect on the migration of the differentiated hNPCs. There was no significant change in the survival of hNPCs after exposure to the pulsed EF. To investigate the role of Ca2+ signaling in electrotactic migration of hNPCs, pharmacological inhibition of Ca2+ channels in the EF-exposed cells revealed that the electrotactic migration of hNPCs exposed to Ca2+ channel blockers was significantly lower compared to the control group. The findings suggest that the pulsed EF induced migration of hNPCs is partly influenced by intracellular Ca2+ signaling.

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.