[Optimized fitting of a midface implant to anchor a magnetic nasal prosthesis using 3D printing]. / 3-D-Druck-optimierte Anpassung eines Mittelgesichtsimplantats zur magnetgetragenen nasalen Epithesenversorgung.

BACKGROUND: Plate-based anchorage systems for craniofacial prostheses offer advantages over extraoral solitary titanium implants in terms of the flexible choice of mounting points and higher stability. Disadvantages become apparent in the complex individual intraoperative adaptation of the plate-based systems to the usually poorly accessible bone. The current article presents a method to overcome these disadvantages and make greater use of the advantages of plate-based systems. MATERIALS AND METHODS: The bony midface of a patient who had undergone rhinectomy for cancer of the nasal entrance was reconstructed as a virtual 3D model based on preoperative CT. The open-source software (3D-Slicer) allowed easy and fast reconstruction as well as adaptation for 3D printing using transparent plastic (MED610; stratasys Ltd., MN, USA). RESULTS: A titanium mini-plate (MEDICON) for anchoring the nasal prosthesis could be fitted extremely precisely on the midface 3D print. Important anatomical structures were spared, and screw placement was selected according to the individual bone thickness. Implantation of the in-advance fitted titanium plate was performed without complications and without further adjustments. CONCLUSION: In-advance fitting of plate-based systems for anchorage of craniofacial prostheses using 3D printing of the midface overcomes their disadvantages of time-consuming and possibly imprecise individual adaptation. This method further exploits the advantages of higher stability through more possible mounting points, even in thinner bone, to prevent loosening. In addition, in-advance fitting of titanium plates on the 3D model enables better identification and protection of important anatomical structures and shortens operative time.

Access to the Apical Cochlear Modiolus for Possible Stem Cell-based and Gene Therapy of the Auditory Nerve.

OBJECTIVE: Loss of spiral ganglion neurons (SGN) is permanent and responsible for a substantial number of patients suffering from hearing impairment. It can derive from the degeneration of SGNs due to the death of sensory hair cells as well as from auditory neuropathy. Utilizing stem cells to recover lost SGNs increasingly emerges as a possible therapeutic option, but access to human SGNs is difficult due to their protected location within the bony impacted cochlea. Aim of this study was to establish a reliable and practicable approach to access SGNs in the human temporal bone for possible stem cell and gene therapies. METHODS: In seven human temporal bone specimen a transcanal approach was used to carefully drill a cochleostomy in the lateral second turn followed by insertion of a tungsten needle into the apical modiolus to indicate the spot for intramodiolar injections. Subsequent cone beam computed tomography (CBCT) served as evaluation for positioning of the marker and cochleostomy size. RESULTS: The apical modiolus could be exposed in all cases by a cochleostomy (1.6 mm2, standard deviation ±0.23 mm2) in the lateral second turn. 3D reconstructions and analysis of CBCT revealed reliable positioning of the marker in the apical modiolus, deviating on average 0.9 mm (standard deviation ±0.49 mm) from the targeted center of the second cochlear turn. CONCLUSION: We established a reliable, minimally invasive, transcanal surgical approach to the apical cochlear modiolus in the human temporal bone in foresight to stem cell-based and gene therapy of the auditory nerve.

Mastoid cavity obliteration and Vibrant Soundbridge implantation for patients with mixed hearing loss.

OBJECTIVES/HYPOTHESIS: To review the results of obliteration of a preexisting mastoid cavity with abdominal fat and Vibrant Soundbridge implantation in patients with mixed hearing loss (MHL) and to compare the data with results of Vibrant Soundbridge implantation in patients with MHL without mastoid cavity and with pure sensorineural hearing loss (SNHL). STUDY DESIGN: Retrospective chart analysis of 10 patients (10 ears) with MHL and preexisting mastoid cavity, 18 patients (19 ears) with MHL alone and nine patients (10 ears) with SNHL treated in one tertiary referral center. METHODS: Vibrant Soundbridge implantation and obliteration in case a mastoid cavity existed previously. Pure tone audiometry (average air-bone gap, average functional gain), speech audiometry (Freiburg Monosyllabic Test) and complication rate were main outcome measures. RESULTS: Postoperative average air-bone gap was -15.1 ± 21.2 dB in patients with MHL with mastoid cavity obliteration, -7.2 ± 11.4 dB in patients with MHL without mastoid cavity, and -5.7 ± 11.2 dB in patients with SNHL. Average functional gain was 40.0 ± 23.5 dB, 39.7 ± 12.1 dB, and 9.5 ± 10.6 dB. Postoperative speech discrimination rate was 77.9 ± 20.8%, 83.3 ± 13.6%, and 83.6 ± 6.3%. No severe intraoperative or postoperative complications were noted. CONCLUSIONS: Mastoid cavity obliteration during Vibrant Soundbridge implantation in patients with MHL and preexisting mastoid cavity is a safe procedure. The audiometric results are satisfying and comparable to those of other patient groups implanted with the same device. LEVEL OF EVIDENCE: 4.

Structure and function of cochlear afferent innervation.

PURPOSE OF REVIEW: For the perception of sound, acoustic signals need to be encoded into a neuronal code. This takes place at the inner hair cells of the organ of Corti and the afferent fibres of the auditory nerve. We will review the current knowledge of the anatomy and function of these elements as well as their connection - formed by the afferent inner hair cell synapse. RECENT FINDINGS: Depending on their tonotopic location, inner hair cells are innervated by 5-30 dendrites of spiral ganglion neurons. Electrophysiological recordings from single fibres demonstrate - apart from a high-frequency selectivity - a pronounced heterogeneity in their response to sound of varying intensity. The source as well as the function of this heterogeneity is not well understood, but recent publications have suggested several mechanisms, including variations in the presynaptic Ca2+ influx and subsequent transmitter release, the postsynaptic sensitivity to neurotransmitter and electrical as well as anatomical variability of single fibres. These mechanisms might act together to expand the dynamic range of sound that can be encoded. SUMMARY: Classical studies as well as recent publications demonstrate that sound encoding at the inner hair cell afferent synapse involves mechanisms leading to tonotopic frequency separation and distribution of intensity coding over many neuronal channels.

The Ca2+ channel subunit beta2 regulates Ca2+ channel abundance and function in inner hair cells and is required for hearing.

Hearing relies on Ca(2+) influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca(2+) channel subunit Ca(V)beta(2) in hearing. Of the Ca(V)beta(1-4) mRNAs, IHCs predominantly contained Ca(V)beta(2). Hearing was severely impaired in mice lacking Ca(V)beta(2) in extracardiac tissues (Ca(V)beta(2)(-/-)). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in Ca(V)beta(2)(-/-) mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. Ca(V)beta(2)(-/-) IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing Ca(V)1.3 channels. Confocal Ca(2+) imaging revealed presynaptic Ca(2+) microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer Ca(V)1.3 channels. The coupling of the remaining Ca(2+) influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the Ca(V)beta(2)(-/-) mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in Ca(V)beta(2)(-/-) mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic Ca(V)1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca(2+) activated potassium channels in IHCs were impaired in the absence of Ca(V)beta(2). Moreover, we found the developmental efferent innervation to persist in Ca(V)beta(2)-deficient IHCs. In summary, Ca(V)beta(2) has an essential role in regulating the abundance and properties of Ca(V)1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.

Tuning of synapse number, structure and function in the cochlea.

Cochlear inner hair cells (IHCs) transmit acoustic information to spiral ganglion neurons through ribbon synapses. Here we have used morphological and physiological techniques to ask whether synaptic mechanisms differ along the tonotopic axis and within IHCs in the mouse cochlea. We show that the number of ribbon synapses per IHC peaks where the cochlea is most sensitive to sound. Exocytosis, measured as membrane capacitance changes, scaled with synapse number when comparing apical and midcochlear IHCs. Synapses were distributed in the subnuclear portion of IHCs. High-resolution imaging of IHC synapses provided insights into presynaptic Ca(2+) channel clusters and Ca(2+) signals, synaptic ribbons and postsynaptic glutamate receptor clusters and revealed subtle differences in their average properties along the tonotopic axis. However, we observed substantial variability for presynaptic Ca(2+) signals, even within individual IHCs, providing a candidate presynaptic mechanism for the divergent dynamics of spiral ganglion neuron spiking.

Conformational switch of syntaxin-1 controls synaptic vesicle fusion.

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells.

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca(v)1.3 voltage-gated Ca(2+) channels. Central to their role in this regard, Ca(v)1.3 channels in inner hair cells show little Ca(2+)-dependent inactivation, a fast negative feedback regulation by incoming Ca(2+) ions, which depends on calmodulin association with the Ca(2+) channel alpha(1) subunit. Ca(2+)-dependent inactivation characterizes nearly all voltage-gated Ca(2+) channels including Ca(v)1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca(v)1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca(2+)-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca(2+) channels. Here, we demonstrate that CaBPs differentially modify Ca(2+) feedback to Ca(v)1.3 channels in transfected cells and explore their significance for Ca(v)1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca(2+)-dependent inactivation of Ca(v)1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca(v)1.3, but CaBP4 more weakly inhibits Ca(2+)-dependent inactivation than CaBP1. Ca(2+)-dependent inactivation is marginally greater in inner hair cells from CaBP4(-/-) than from wild-type mice, yet CaBP4(-/-) mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4(-/-) mice and therefore is positioned to modulate native Ca(v)1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca(2+) channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca(v)1.3 Ca(2+) currents required for auditory transmission.

A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis.

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1.

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.