The effects of mild hypothermia on the electrode insertion trauma in a murine whole organ cochlea culture.

Introduction: Local therapeutic hypothermia (32°C) has been linked experimentally to an otoprotective effect in the electrode insertion trauma. The pathomechanism of the electrode insertion trauma is connected to the activation of apoptosis and necrosis pathways, pro-inflammatory and fibrotic mechanisms. In a whole organ cochlea culture setting the effect of therapeutic hypothermia in an electrode insertion trauma model is evaluated. Material and Methods: The cochleae of C57Bl6/J mice (Charles River®, Freiburg, Germany) are cultured for 24 hours at 37°C and 32°C after inserting a fishing line through the round window simulating an insertion trauma. The resulting effect was evaluated for the apoptotic reaction - B-cell-Lymphoma-2-Associated-X-Protein (BAX), B-Cell-Lymphoma-2-Protein (BCL2) and Cleaved-Caspase-3 (CC3) -, the inflammatory response - Tumor-Necrosis-Factor-Alpha (TNFα), Interleukin-1-Beta (IL-1Imm) and Cyclooxygenase-2 (COX2) - and proliferation process - Transforming-Growth-Factor-Beta-1 (TGFß1) - using immunohistochemistry and real-time PCR technique. A minimum of 12 cochlea per experiment were used. Results: A pro-apoptotic situation was observed in the normothermic group (BAX, CC3 ˃ Bcl2) whereas an anti-apoptotic constellation was found at 32°C culture conditions (BAX, CC3 < Bcl2). Furthermore the effect of the IT knowing to effect the pro-inflammatory cytokine (TNFα, Il1ß) and enzyme (COX2) expression has been reproduced. This reaction was reversed with the application of therapeutic hypothermia resulting in significant lower pro-inflammatory cytokine (TNFα, Il1ß) and enzyme (COX2) expression. TGFß1 was increased by hypothermia. Discussion: Concluding a protective effect of hypothermia on the experimental electrode insertion trauma can be described by an anti-apoptotic and anti-inflammatory reaction.

Drafting a Surgical Procedure Using a Computational Anatomy Driven Approach for Precise, Robust, and Safe Vestibular Neuroprosthesis Placement-When One Size Does Not Fit All.

OBJECTIVE: To design and evaluate a new vestibular implant and surgical procedure that should reach correct electrode placement in 95% of patients in silico. DESIGN: Computational anatomy driven implant and surgery design study. SETTING: Tertiary referral center. PARTICIPANTS: The population comprised 81 patients that had undergone a CT scan of the Mastoid region in the Maastricht University Medical Center. The population was subdivided in a vestibular implant eligible group (28) and a control group (53) without known vestibular loss. INTERVENTIONS: Canal lengths and relationships between landmarks were calculated for every patient. The relationships in group-anatomy were used to model a fenestration site on all three semicircular canals. Each patient's simulated individual distance from the fenestration site to the ampulla was calculated and compared with the populations average to determine if placement would be successful. MAIN OUTCOME MEASURES: Lengths of the semicircular canals, distances from fenestration site to ampulla (intralabyrinthine electrode length), and rate of successful electrode placement (robustness). RESULTS: The canal lengths for the lateral, posterior, and superior canal were respectively 12.1 mm ±â€Š1.07, 18.8 mm ±â€Š1.62, and 17.5 mm ±â€Š1.23, the distances from electrode fenestration site to the ampulla were respectively 3.73 mm ±â€Š0.53, 9.02 mm ±â€Š0.90, and 5.31 mm ±â€Š0.73 and electrode insertions were successful for each respective semicircular canal in 92.6%, 66.7%, and 86.4% of insertions in silico. The implant electrode was subsequently revised to include two more electrodes per lead, resulting in a robustness of 100%. CONCLUSIONS: The computational anatomy approach can be used to design and test surgical procedures. With small changes in electrode design, the proposed surgical procedure's target robustness was reached.

Expression of trans-membrane serine protease 3 (TMPRSS3) in the human organ of Corti.

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.

The Human "Cochlear Battery" - Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea.

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.

Supernumerary human hair cells-signs of regeneration or impaired development? A field emission scanning electron microscopy study.

BACKGROUND: Current attempts to regenerate cochlear sensorineural structures motivate further inspection of the human organ of hearing. Here, we analyzed the supernumerary inner hair cell (sIHC), a possible sign of regeneration and cell replacement. METHODS: Human cochleae were studied using field emission scanning electron microscopy (FESEM; maximum resolution 2 nm) obtained from individuals aged 44, 48, and 58 years with normal sensorineural pure-tone average (PTA) thresholds (PTA <20 dB). The wasted tissue was harvested during trans-cochlear approaches and immediately fixed for ultrastructural analysis. RESULTS: All specimens exhibited sIHCs at all turns except at the extreme lower basal turn. In one specimen, it was possible to image and count the inner hair cells (IHCs) along the cochlea representing the 0.2 kHz-8 kHz region according to the Greenwood place/frequency scale. In a region with 2,321 IHCs, there were 120 scattered one-cell losses or 'gaps' (5%). Forty-two sIHCs were present facing the modiolus. Thirty-eight percent of the sIHCs were located near a 'gap' in the IHC row (±6 IHCs). CONCLUSIONS: The prevalence of ectopic inner hair cells was higher than expected. The morphology and placement could reflect a certain ongoing regeneration. Further molecular studies are needed to verify if the regenerative capacity of the human auditory periphery might have been underestimated.

Irradiation as a hazard for mucociliary clearance.

OBJECTIVES: In this paper we study effects of irradiation to pulmonary tissue on a micro and ultrastructural level to get insights into the dynamics of morphological changes and associated post-radiative physiological conditions. METHODS: Animal and human pulmonary tissue with and without radiation damage was subject to light, transmission, scanning and polarization microscopy and morphometric evaluation. RESULTS: The present investigations on the influence of irradiation on experimental and human lung tissue demonstrate that complex changes are induced in the cells which are essential for mucociliary clearance. These changes are a shortage of alveolar macrophages, cell apoptosis, proliferation of collagen ligament in the barrier of gaseous exchange, retraction of endothelial lining of capillaries and significant broadening of the gaseous exchange barrier, resulting in serious damage for the O2 and CO2 exchange. CONCLUSIONS: These changes at microscopic, cellular, and ciliary level trigger conditions for various diseases of the respiratory system, which is further assessed by a simultaneous computer aided estimation of ciliary function. With the concurrent world-wide increase of respiratory diseases, these findings are important knowledge for the clinical practice.

Molecular organization and fine structure of the human tectorial membrane: is it replenished?

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.

Macromolecular organization and fine structure of the human basilar membrane - RELEVANCE for cochlear implantation.

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 electron microscopy (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-ß2 and collagen IV, (2) BM "proper" 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 µm). 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.

Human cochlea: anatomical characteristics and their relevance for cochlear implantation.

This is a review of the anatomical characteristics of human cochlea and the importance of variations in this anatomy to the process of cochlear implantation (CI). Studies of the human cochlea are essential to better comprehend the physiology and pathology of man's hearing. The human cochlea is difficult to explore due to its vulnerability and bordering capsule. Inner ear tissue undergoes quick autolytic changes making investigations of autopsy material difficult, even though excellent results have been presented over time. Important issues today are novel inner ear therapies including CI and new approaches for inner ear pharmacological treatments. Inner ear surgery is now a reality, and technical advancements in the design of electrode arrays and surgical approaches allow preservation of remaining structure/function in most cases. Surgeons should aim to conserve cochlear structures for future potential stem cell and gene therapies. Renewal interest of round window approaches necessitates further acquaintance of this complex anatomy and its variations. Rough cochleostomy drilling at the intricate "hook" region can generate intracochlear bone-dust-inducing fibrosis and new bone formation, which could negatively influence auditory nerve responses at a later time point. Here, we present macro- and microanatomic investigations of the human cochlea viewing the extensive anatomic variations that influence electrode insertion. In addition, electron microscopic (TEM and SEM) and immunohistochemical results, based on specimens removed at surgeries for life-threatening petroclival meningioma and some well-preserved postmortal tissues, are displayed. These give us new information about structure as well as protein and molecular expression in man. Our aim was not to formulate a complete description of the complex human anatomy but to focus on aspects clinically relevant for electric stimulation, predominantly, the sensory targets, and how surgical atraumaticity best could be reached.

Distribution of P75 neurotrophin receptor in adult human cochlea--an immunohistochemical study.

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.

The cochlea in fetuses with neural tube defects.

In this study different malformations of the cochlea could be demonstrated. Nevertheless, we could not delineate a distinct malformation of the inner ear, that can be linked to a neural tube defect. Neural tube defects are a frequent and heterogeneous group of malformations, ranging from the survivable spina bifida to fatal anencephaly. In multiple animal models an involvement of the vestibulocochlear system has been demonstrated. In this article human fetal temporal bones of neural tube defects were analysed in a multimodular work-up. The morphologic study was performed with light microscopy, transmission electron microscopy and synchrotron radiation-based microcomputed tomography. Immunohistochemistry for different neuronal markers such as peripherin, beta-III-tubulin and vimentin helped to evaluate ontogenetic tissue development. Eight fetal temporal bones with neural tube defects and five control temporal bones were included into the morphologic study. The morphologic results of the neural tube defect temporal bones showed six regularly developed cochleas and two with only a single cochlear turn. Three of the neural tube defect temporal bones were further examined with immunohistochemical analysis. No differences in the staining pattern for peripherin, beta-III-tubulin and vimentin were detected.

Epidermal growth factor receptor expression in human fetal cochlea with Turner syndrome.

HYPOTHESIS: Growth hormones have beneficial effects on increasing height in adults with Turner syndrome (TS) and may also affect auditory function. BACKGROUND: Turner syndrome is the most common sex-linked chromosomal abnormality in female conceptions. Epidermal growth factor and its receptor (EGFR) affect differentiation, proliferation, and migration of epithelial cells and function as survival factors. The expression of EGFR is found in the developing and juvenile inner ear of experimental animals but is absent in adults. METHODS: To determine whether EGFR plays a role in TS, its expression was analyzed in the cochlea of healthy fetus and fetus with TS and in healthy adults. RESULTS: In healthy fetuses, EGFR protein expression was localized to the inner and outer hair cells and the Reissner membrane. The fetuses with TS on the 13th gestational week (GW) showed a similar pattern of immunoreactivity as the normal 16th and 20th GW cochlea. By the 23rd GW, EGFR immunoreactivity was not detectable in the TS hair cells or the Reissner membrane, and less intensive staining was found in the surrounding fibrocytes of the spiral ganglion. CONCLUSION: This is the first demonstration of EGFR immunoreactivity in the human cochlea and illustrates how EGFR expression is altered during development in TS. These findings indicate the importance of growth hormone receptors for inner ear development in humans.

Does perinatal asphyxia induce apoptosis in the inner ear?

Pre- and perinatal asphyxia is known to be an important risk factor in the development of neonatal hearing impairment. This study aims to evaluate the role of apoptosis, which is known to play an essential role in the development of the inner ear structures, in the development of neonatal hearing loss caused by pre- and perinatal asphyxia. Eight temporal bones of six different newborns were included. We performed a morphologic analysis by both light microscopy, and transmission electron microscopy, as well as immunohistochemical staining to detect the cleaved form of caspase 3 as apoptosis marker and Bcl 2 as anti-apoptotic marker. Early and late phases of apoptosis were evidenced by condensation of chromatin (electron-dense, black structure along nuclear membrane) and fragmentation of the nucleus, respectively. Changes in nuclear morphology during apoptosis correlate with cleavage by caspase 3 located downstream of Bcl 2 action. The immunohistochemistry for cleaved caspase 3 showed a particular predilection for the inner and outer hair cells, spiral ganglion cells and the marginal cells of the stria vascularis. The brain of all examined cases did not show signs of apoptosis. In summary, this investigation suggests that apoptosis takes place before brain tissue apoptosis and is probably an earlier event than thought. Apoptosis of the cochlea is known to play an essential role in the development of the inner ear. Additionally, this study shows that apoptosis may play an important role in the development of hearing impairment, caused by pre- and perinatal asphyxia.

Phenotypic characterization and distribution of dendritic cells in parotid gland tumors.

Evidence on the relationship between the infiltration of dendritic cells (DCs) and prognosis in head and neck tumors exists. Interestingly, only limited information is available regarding the maturation state and distribution of DCs in parotid gland tumors. The purpose of our study was therefore to extend these observations and to investigate in more detail the density and distribution of mature DCs and Langerhans cells (LCs) in parotid gland tumors. We present immunohistochemical evidence of characterization and distribution of DCs and LCs in parotid gland tumors, enclosed pleomorphic adenomas and malignant parotid tumors. Two populations of mature DCs could be identified, P55(+)-DCs and DC-LAMP(+)-DCs, whereas LCs could be identified as Langerin(+)-LCs. The overall impression was that parotid gland tumors contained only few mature DCs and LCs. Considering the sparsity of mature DCs in the malignant tissues, anti-tumor response can be only limited. On the basis of our data, we imply that the application of DC vaccination in combination with other modalities for treatment of parotid gland carcinoma should be taken into account. In this regard, the utilization of DC immunotherapy for management of minimal residual disease after resection of primary tumor can be promising. Putative targets expressed in this type of tumors have to be defined.