Immuno-surveillance and protection of the human cochlea.

Background: Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Materials and methods: Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Results: Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. Discussion: The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

Distribution of Immune Cells Including Macrophages in the Human Cochlea.

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression. Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization. Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling "antennae" projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti. Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

"Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

The human endolymphatic sac (ES) is believed to regulate inner ear fluid homeostasis and to be associated with Meniere's disease (MD). We analyzed the ion transport protein sodium/potassium-ATPase (Na/K-ATPase) and its isoforms in the human ES using super-resolution structured illumination microscopy (SR-SIM). Human vestibular aqueducts were collected during trans-labyrinthine vestibular schwannoma surgery after obtaining ethical permission. Antibodies against various isoforms of Na/K-ATPase and additional solute-transporting proteins, believed to be essential for ion and fluid transport, were used for immunohistochemistry. A population of epithelial cells of the human ES strongly expressed Na/K-ATPase α1, ß1, and ß3 subunit isoforms in either the lateral/basolateral or apical plasma membrane domains. The ß1 isoform was expressed in the lateral/basolateral plasma membranes in mostly large cylindrical cells, while ß3 and α1 both were expressed with "reversed polarity" in the apical cell membrane in lower epithelial cells. The heterogeneous expression of Na/K-ATPase subunits substantiates earlier notions that the ES is a dynamic structure where epithelial cells show inverted epithelial transport. Dual absorption and secretion processes may regulate and maintain inner ear fluid homeostasis. These findings may shed new light on the etiology of endolymphatic hydrops and MD.

Expression of Na/K-ATPase subunits in the human cochlea: a confocal and super-resolution microscopy study with special reference to auditory nerve excitation and cochlear implantation.

Background: For the first time the expression of the ion transport protein sodium/potassium-ATPase and its isoforms was analyzed in the human cochlea using light- and confocal microscopy as well as super-resolution structured illumination microscopy. It may increase our understanding of its role in the propagation and processing of action potentials in the human auditory nerve and how electric nerve responses are elicited from auditory prostheses. Material and methods: Archival human cochlear sections were obtained from trans-cochlear surgeries. Antibodies against the Na/K-ATPase ß1 isoform together with α1 and α3 were used for immunohistochemistry. An algorithm was applied to assess the expression in various domains. Results: Na/K ATPase ß1 subunit was expressed, mostly combined with the α1 isoform. Neurons expressed the ß1 subunit combined with α3, while satellite glial cells expressed the α1 isoform without recognized association with ß1. Types I and II spiral ganglion neurons and efferent fibers expressed the Na/K-ATPase α3 subunit. Inner hair cells, nerve fibers underneath, and efferent and afferent fibers in the organ of Corti also expressed α1. The highest activity of Na/K-ATPase ß1 was at the inner hair cell/nerve junction and spiral prominence. Conclusion: The human auditory nerve displays distinct morphologic features represented in its molecular expression. It was found that electric signals generated via hair cells may not go uninterrupted across the spiral ganglion, but are locally processed. This may be related to particular filtering properties in the human acoustic pathway.

Human Inner Ear Immune Activity: A Super-Resolution Immunohistochemistry Study.

Background: Like the brain, the human inner ear was long thought to be devoid of immune activity. Only the endolymphatic sac (ES) was known to be endowed with white blood cells that could process antigens and serve as an immunologic defense organ for the entire inner ear. Unexpectedly, the cochlear and vestibular organs, including the eighth cranial nerve, were recently shown to contain macrophages whose functions and implication in ear disease are somewhat undefined. Here, we review recent inner ear findings in man and extend the analyses to the vestibular nerve using super-resolution structured illumination microscopy (SR-SIM). Materials and Methods: Human ESs and cochleae were collected during surgery to treat patients with vestibular schwannoma and life-threatening petro-clival meningioma compressing the brainstem. The ESs and cochleae were placed in fixative, decalcified, and rapidly frozen and cryostat sectioned. Antibodies against ionized calcium-binding adaptor molecule 1-expressing cells (IBA1 cells), laminin ß2 and type IV collagen TUJ1, cytokine fractalkine (CX3CL1), toll-like receptor 4 (TLR4), CD68, CD11b, CD4, CD8, the major histocompatibility complex type II (MHCII), and the microglial marker TEME119 were used. Results: IBA1-positive cells were present in the ESs, the cochlea, central and peripheral axons of the cochlear nerve, and the vestibular nerve trunk. IBA1 cells were found in the cochlear lateral wall, spiral limbus, and spiral ganglion. Notable variants of IBA1 cells adhered to neurons with "synapse-like" specializations and cytoplasmic projections. Slender IBA1 cells occasionally protracted into the basal lamina of the Schwann cells and had intimate contact with surrounding axons. Discussion: The human eighth nerve may be under the control of a well-developed macrophage cell system. A small number of CD4+ and CD8+ cells were found in the ES and occasionally in the cochlea, mostly located in the peripheral region of Rosenthal's canal. A neuro-immunologic axis may exist in the human inner ear that could play a role in the protection of the auditory nerve. The implication of the macrophage system during disease, surgical interventions, and cell-based transplantation should be further explored.

The Human Endolymphatic Sac and Inner Ear Immunity: Macrophage Interaction and Molecular Expression.

Background: The endolymphatic sac (ES) is endowed with a multitude of white blood cells that may trap and process antigens that reach the inner ear from nearby infection-prone areas, it thus serves as an immunologic defense organ. The human ES, and unexpectedly the rest of the inner ear, has been recently shown to contain numerous resident macrophages. In this paper, we describe ES macrophages using super-resolution structured fluorescence microscopy (SR-SIM) and speculate on these macrophages' roles in human inner ear defense. Material and Methods: After ethical permission was obtained, human vestibular aqueducts were collected during trans-labyrinthine surgery for acoustic neuroma removal. Tissues were placed in fixative before being decalcified, rapidly frozen, and cryostat sectioned. Antibodies against IBA1, cytokine fractalkine (CX3CL1), toll-like receptor 4 (TLR4), cluster of differentiation (CD)68, CD11b, CD4, CD8, and the major histocompatibility complex type II (MHCII) were used for immunohistochemistry. Results: A large number of IBA1-positive cells with different morphologies were found to reside in the ES; the cells populated surrounding connective tissue and the epithelium. Macrophages interacted with other cells, showed migrant behavior, and expressed immune cell markers, all of which suggest their active role in the innate and adaptive inner ear defense and tolerance. Discussion: High-resolution immunohistochemistry shows that antigens reaching the ear may be trapped and processed by an immune cell machinery located in the ES. Thereby inflammatory activity may be evaded near the vulnerable inner ear sensory structures. We speculate on the immune defensive link between the ES and the rest of the inner ear.

Guided growth of auditory neurons: Bioactive particles towards gapless neural - electrode interface.

Cochlear implant (CI) is a successful device to restore hearing. Despite continuous development, frequency discrimination is poor in CI users due to an anatomical gap between the auditory neurons and CI electrode causing current spread and unspecific neural stimulation. One strategy to close this anatomical gap is guiding the growth of neuron dendrites closer to CI electrodes through targeted slow release of neurotrophins. Biodegradable calcium phosphate hollow nanospheres (CPHSs) were produced and their capacity for uptake and release of neurotrophins investigated using 125I-conjugated glia cell line-derived neurotrophic factor (GDNF). The CPHSs were coated onto CI electrodes and loaded with neurotrophins. Axon guidance effect of slow-released neurotrophins from the CPHSs was studied in an in vitro 3D culture model. CPHS coating bound and released GDNF with an association rate constant 6.3 × 103 M-1s-1 and dissociation rate 2.6 × 10-5 s-1, respectively. Neurites from human vestibulocochlear ganglion explants found and established physical contact with the GDNF-loaded CPHS coating on the CI electrodes placed 0.7 mm away. Our results suggest that neurotrophin delivery through CPHS coating is a plausible way to close the anatomical gap between auditory neurons and electrodes. By overcoming this gap, selective neural activation and the fine hearing for CI users become possible.

Taste disturbance after stapes surgery: an evaluation of frequency, severity, duration, and quality-of-life.

CONCLUSION: The incidence of taste disturbance after stapes surgery is high (61.9%), whereas the majority (94.8%) recovers within 1 year. More severe surgical nerve trauma caused more disturbance, implying that the nerve should be handled carefully during surgery. OBJECTIVES: Patients operated on for otosclerosis seem more often to complain about post-operative taste disturbance than those operated on for chronic otitis media, although the chorda tympani nerve more seldom becomes maltreated in stapedotomy. These observations seem paradoxical. It is unclear to what extent a post-operative taste disturbance affects the quality-of-life. This study aims to shed light on the occurrence of post-operative taste disturbances, on possible prognostic factors, and to what extent post-operative taste disturbance impairs the quality-of-life. METHODS: One hundred and thirty-four adults undergoing primary stapedotomy were included. Questionnaires on taste disturbance and quality-of-life (SF-36) were answered before and after surgery, until 1 year post-operatively. RESULTS: Eighty-three (61.9%) study persons reported post-operative taste disturbance. Seven (5.2%) reported persisting disturbance at 1 year. Surgically more traumatized chorda tympani nerves correlated with more severe taste disturbance post-operatively than less traumatized. Taste disturbance at 1 year post-operatively correlate with a decrease of the physical function domain in the SF-36.

High-frequency hearing, tinnitus, and patient satisfaction with stapedotomy: A randomized prospective study.

Otosclerosis is a common disorder that leads to conductive hearing loss. Most patients with otosclerosis also have tinnitus, and surgical treatment is known to improve hearing as well as tinnitus. Some patients however experience worsening of tinnitus after the operation, but there are no known factors that allow surgeons to predict who will be at risk. In this prospective observational study on 133 patients undergoing stapedotomy, we show that postoperative air conduction thresholds at very high stimulus frequencies predict improvement of tinnitus, as assessed with proportional odds logistic regression models. Young patients were significantly more likely to experience reduction of tinnitus and patients whose tinnitus became better were also more satisfied with the outcome of the operation. These findings have practical importance for patients and their surgeons. Young patients can be advised that surgery is likely to be beneficial for their tinnitus, but a less positive message should be conveyed to older patients.

A randomised, double blind trial of N-Acetylcysteine for hearing protection during stapes surgery.

BACKGROUND: Otosclerosis is a disorder that impairs middle ear function, leading to conductive hearing loss. Surgical treatment results in large improvement of hearing at low sound frequencies, but high-frequency hearing often suffers. A likely reason for this is that inner ear sensory cells are damaged by surgical trauma and loud sounds generated during the operation. Animal studies have shown that antioxidants such as N-Acetylcysteine can protect the inner ear from noise, surgical trauma, and some ototoxic substances, but it is not known if this works in humans. This trial was performed to determine whether antioxidants improve surgical results at high frequencies. METHODS: We performed a randomized, double-blind and placebo-controlled parallel group clinical trial at three Swedish university clinics. Using block-stratified randomization, 156 adult patients undergoing stapedotomy were assigned to intravenous N-Acetylcysteine (150 mg/kg body weight) or matching placebo (1:1 ratio), starting one hour before surgery. The primary outcome was the hearing threshold at 6 and 8 kHz; secondary outcomes included the severity of tinnitus and vertigo. FINDINGS: One year after surgery, high-frequency hearing had improved 2.7 ± 3.8 dB in the placebo group (67 patients analysed) and 2.4 ± 3.7 dB in the treated group (72 patients; means ± 95% confidence interval, p = 0.54; linear mixed model). Surgery improved tinnitus, but there was no significant intergroup difference. Post-operative balance disturbance was common but improved during the first year, without significant difference between groups. Four patients receiving N-Acetylcysteine experienced mild side effects such as nausea and vomiting. CONCLUSIONS: N-Acetylcysteine has no effect on hearing thresholds, tinnitus, or balance disturbance after stapedotomy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00525551.

The chorda tympani degenerates during chronic otitis media: an electron microscopy study.

CONCLUSION: Chorda tympani nerve specimens from ears with chronic inflammatory middle ear disease exhibit structural signs of degeneration. These correlate well with taste disturbance. Simultaneously, they exhibit signs of regeneration, which may explain the ability for taste recovery. OBJECTIVES: The chorda tympani, the major taste nerve, runs uncovered through the middle ear cavity. This situation exposes it to various forms of middle ear pathology. A difference has been noticed regarding taste symptoms pre- and postoperatively between inflammatory and non-inflammatory diseases. The present study aimed to investigate ultrastructural changes of chorda tympani in different forms of inflammatory middle ear disease, such as chronic suppurative otitis media and cholesteatoma, as compared with normal. METHODS: Five chorda tympani specimens were collected from healthy middle ears of patients subjected to surgery for acoustic neuroma, to be used as normal controls, and five from middle ears with chronic otitis media or cholesteatoma, where the nerve could not be saved during the operation. Light and electron microscopy were performed. RESULTS: For all five nerves from diseased ears, microscopy showed a higher percentage of axon and myelin sheath degeneration than in the normal controls. Furthermore, three of the five also exhibited sprouting.

Non-echo planar diffusion-weighted MRI increases follow-up accuracy after one-step step canal wall-down obliteration surgery for cholesteatoma.

CONCLUSION: Non-echo planar (non-EPI) diffusion-weighted (DW) magnetic resonance imaging (MRI) increases the number of detected cholesteatoma after one-step canal wall-down (CWD) obliteration surgery for cholesteatoma compared with clinical evaluation alone. OBJECTIVE: To evaluate the use of DW-MRI for detection of cholesteatoma after surgical treatment using a CWD obliteration technique. METHODS: Thirty-eight adult patients (41 ears) treated with an identical one-step CWD obliteration surgical technique were included in a prospective and blinded study. All patients were investigated with non-EPI and EPI DW-MRI 1-9 months after the clinical examination. Follow-up time after primary surgery varied between 10 and 234 months. DW-MRI was assessed by two neuroradiologists and compared with clinical results. Inter-rater agreement was calculated. Positive non-EPI DW-MRI cases underwent revision surgery within 18-159 days after imaging. RESULTS: Seven of 41 cases were evaluated as positive for cholesteatoma on non-EPI DW-MRI. Since one patient refused surgery six of these seven cases underwent surgical revision and all were verified. There was agreement between clinical and non-EPI findings in five of eight cases. EPI findings correlated poorly with non-EPI and clinical findings. Inter-rater agreement (Cohen's kappa) was 0.91 for non-EPI DW-MRI (p < 0.001) and -0.062 for EPI DW-MRI (p = 0.43).

Surgical treatment of patients with facial neuromas--a report of 26 consecutive operations.

OBJECTIVE: To analyze surgical treatment and outcome in patients with facial neuromas at a tertiary referral hospital. STUDY DESIGN: A chart review of 26 patients treated between 1971 and 2006, with questionnaire follow-up ranging from 2 to 19 years. All patients except one were operated with radical tumor removal approaches. RESULTS: Approximately 54% of the patients presented with symptoms related to the VIIth cranial nerve (facial palsy and facial spasm), 58% with symptoms related to the VIIIth cranial nerve (hearing deficit, tinnitus, and vertigo), and 8% related to the Vth cranial nerve (facial pain and facial sensory deficit). Approximately 39% presented with no facial symptoms. Twenty-one patients received a facial nerve graft from the greater auricular nerve or the sural nerve; 1 patient had an accessory-facial anastomosis. One patient had a subtotal tumor removal preserving the facial nerve. Three patients were not grafted. Most tumors (88%) affect the geniculate ganglion. Approximately 82% of the grafted patients regained a House-Brackmann facial nerve function (HB) grade III; 14% regained HB grades IV to V. No serious morbidity or mortality was reported. No recurrences have been reported where a total tumor removal was performed. CONCLUSION: Surgical removal of facial neuroma is a safe procedure with a low complication rate and a low recurrence rate. First symptoms are diverse and are predominantly derived from the facial and vestibulocochlear nerve. Facial nerve grafting is reliable, giving the patient an acceptable facial nerve function (HB III).